Genetically modified major histocompatibility complex mice

ABSTRACT

The invention provides genetically modified non-human animals that express chimeric human/non-human MHC I polypeptide and/or human or humanized β2 microglobulin polypeptide, as well as embryos, cells, and tissues comprising the same. Also provided are constructs for making said genetically modified animals and methods of making the same. Methods of using the genetically modified animals to study various aspects of human immune system are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. Provisional PatentApplication Nos. 61/552,582 and 61/552,587, both filed Oct. 28, 2011,and U.S. Provisional Patent Application No, 61/700,908, filed Sep. 14,2012, all of which are hereby incorporated by reference herein in theirentireties.

FIELD OF THE INVENTION

Present invention relates to a genetically modified non-human animal,e.g., a rodent (e.g., a mouse or a rat), that expresses a human orhumanized Major Histocompatibility Complex (MHC) class I molecule. Theinvention also relates to a genetically modified non-human animal, e.g.,a mouse or a rat, that expresses a human or humanized MHC I protein(e.g., MHC I α chain) and/or a human or humanized β2 microglobulin; aswell as embryos, tissues, and cells expressing the same. The inventionfurther provides methods for making a genetically modified non-humananimal that expresses human or humanized MHC class I protein (e.g., MHCI α chain) and/or β2 microglobulin. Also provided are methods foridentifying and evaluating peptides in the context of a humanizedcellular immune system in vitro or in a genetically modified non-humananimal, and methods of modifying an MHC I and/or a β2 microglobulinlocus of a non-human animal, e.g., a mouse or a rat, to express a humanor humanized MHC I and/or β2 microglobulin.

BACKGROUND OF THE INVENTION

In the adaptive immune response, foreign antigens are recognized byreceptor molecules on B lymphocytes (e.g., immunoglobulins) and Tlymphocytes (e.g., T cell receptor or TCR). These foreign antigens arepresented on the surface of cells as peptide fragments by specializedproteins, generically referred to as major histocompatibility complex(MHC) molecules. MHC molecules are encoded by multiple loci that arefound as a linked cluster of genes that spans about 4 Mb. In mice, theMHC genes are found on chromosome 17, and for historical reasons arereferred to as the histocompatibility 2 (H-2) genes. In humans, thegenes are found on chromosome 6 and are called human leukocyte antigen(HLA) genes. The loci in mice and humans are polygenic; they includethree highly polymorphic classes of MHC genes (class I, II and III) thatexhibit similar organization in human and murine genomes (see FIG. 2 andFIG. 3, respectively).

MHC loci exhibit the highest polymorphism in the genome: some genes arerepresented by >300 alleles (e.g., human HLA-DRβ and human HLA-B). Allclass I and II MHC genes can present peptide fragments, but each geneexpresses a protein with different binding characteristics, reflectingpolymorphisms and allelic variants. Any given individual has a uniquerange of peptide fragments that can be presented on the cell surface toB and T cells in the course of an immune response.

Both humans and mice have class I MHC genes (see FIG. 2 and FIG. 3). Inhumans, the classical class I genes are termed HLA-A, HLA-B and HLA-C,whereas in mice they are H-2K, H-2D and H-2L. Class I molecules consistof two chains: a polymorphic α-chain (sometimes referred to as heavychain) and a smaller chain called β2-microglobulin (also known as lightchain), which is generally not polymorphic (FIG. 1). These two chainsform a non-covalent heterodimer on the cell surface. The α-chaincontains three domains (α1, α2 and α3). Exon 1 of the α-chain geneencodes the leader sequence, exons 2 and 3 encode the α1 and α2 domains,exon 4 encodes the α3 domain, exon 5 encodes the transmembrane domain,and exons 6 and 7 encode the cytoplasmic tail. The α-chain forms apeptide-binding cleft involving the α1 and α2 domains (which resembleIg-like domains) followed by the α3 domain, which is similar toβ2-microglobulin.

β2 microglobulin is a non-glycosylated 12 kDa protein; one of itsfunctions is to stabilize the MHC class I α-chain. Unlike the α-chain,the β2 microglobulin does not span the membrane. The human β2microglobulin locus is on chromosome 15, while the mouse locus is onchromosome 2, β32 microglobulin gene consists of 4 exons and 3 introns.Circulating forms of β2 microglobulin are present in the serum, urine,and other body fluids; thus, the non-covalently MHC I-associated β2microglobulin can be exchanged with circulating β2 microglobulin underphysiological conditions.

Class I MHC molecules are expressed on all nucleated cells, includingtumor cells. They are expressed specifically on T and B lymphocytes,macrophages, dendritic cells and neutrophils, among other cells, andfunction to display peptide fragments (typically 8-10 amino acids inlength) on the surface to CD8+ cytotoxic T lymphocytes (CTLs). CTLs arespecialized to kill any cell that bears an MHC I-bound peptiderecognized by its own membrane-bound TCR. When a cell displays peptidesderived from cellular proteins not normally present (e.g., of viral,tumor, or other non-self origin), such peptides are recognized by CTLs,which become activated and kill the cell displaying the peptide.

Typically, presentation of normal (i.e., self) proteins in the contextof MHC I molecules does not elicit CTL activation due to the tolerancemechanisms. However, in some diseases (e.g., cancer, autoimmunediseases) peptides derived from self-proteins become a target of thecellular component of the immune system, which results in destruction ofcells presenting such peptides. Although there has been advancement inrecognizing some self-derived antigens that elicit cellular immuneresponse (e.g., antigens associated with various cancers), in order toimprove identification of peptides recognized by human CTLs through MHCclass I molecules there remains a need for both in vivo and in vitrosystems that mimic aspects of the human cellular immune system. Systemsthat mimic the human cellular immune system can be used in identifyingdisease-associated antigens in order to develop human therapeutics,e.g., vaccines and other biologics. Systems for assessing antigenrecognition in the context of the human immune system can assist inidentifying therapeutically useful CTL populations (e.g., useful forstudying and combatting human disease). Such systems can also assist inenhancing the activity of human CTL populations to more effectivelycombat infections and foreign antigen-bearing entities. Thus, there is aneed for biological systems (e.g., genetically engineered animals) thatcan generate an immune system that displays components that mimic thefunction of human immune system.

SUMMARY OF THE INVENTION

A biological system for generating or identifying peptides thatassociate with human MHC class I proteins and chimeras thereof, and bindto CD8+ T cells, is provided. Non-human animals comprising non-humancells that express human or humanized molecules that function in thecellular immune response are provided. Humanized rodent loci that encodehuman or human or humanized MHC I and β2 microglobulin proteins are alsoprovided. Humanized rodent cells that express human or humanized MHC andβ2 microglobulin molecules are also provided. In vivo and in vitrosystems are provided that comprise humanized rodent cells, wherein therodent cells express one or more human or humanized immune systemmolecules.

Provided herein is a non-human animal, e.g., a rodent (e.g., a mouse ora rat), comprising in its genome a nucleotide sequence encoding achimeric human/non-human (e.g., human/rodent, e.g., human/mouse orhuman/rat) MHC I polypeptide, wherein a human portion of the chimericpolypeptide comprises an extracellular domain of a human MHC Ipolypeptide. Specifically, provided herein is a non-human animalcomprising at an endogenous MHC I locus a nucleotide sequence encoding achimeric human/non-human MHC I polypeptide, wherein a human portion ofthe chimeric polypeptide comprises an extracellular domain of a humanMHC I polypeptide, and wherein the animal expresses the chimerichuman/non-human MHC I polypeptide. In one aspect, the animal does notexpress an extracellular domain of an endogenous non-human MHC Ipolypeptide from an endogenous non-human MHC I locus. In one aspect ofthe invention, the non-human animal (e.g., a rodent, e.g., a mouse or arat) comprises two copies of the MHC I locus comprising a nucleotidesequence encoding chimeric human/non-human (e.g., human/rodent, e.g.,human/mouse or human/rat) MHC I polypeptide. In another aspect of theinvention, the animal comprises one copy of the MHC I locus comprising anucleotide sequence encoding a chimeric human/non-human MHC Ipolypeptide. Thus, the animal may be homozygous or heterozygous for theMHC I locus comprising a nucleotide sequence encoding chimerichuman/non-human MHC I polypeptide. In various embodiments, thenucleotide sequence encoding a chimeric human/non-human MHC Ipolypeptide is comprised in the germline of the non-human animal (e.g.,rodent, e.g., rat or mouse).

In one aspect, the nucleotide sequence encoding the chimerichuman/non-human MHC I is operably linked to endogenous non-humanregulatory elements, e.g., promoter, enhancer, silencer, etc. In oneembodiment, a human portion of the chimeric polypeptide comprises ahuman leader sequence. In an additional embodiment, the human portion ofthe chimeric polypeptide comprises α1, α2, and α3 domains of the humanMHC I polypeptide. The human MHC I polypeptide may be selected from agroup consisting of HLA-A, HLA-B, and HLA-C. In one embodiment, thehuman MHC I polypeptide is an HLA-A2 polypeptide, e.g., an HLA-A2.1polypeptide.

In one aspect, the genetically engineered non-human animal is a rodent.In one embodiment, the rodent is a mouse. Thus, in one embodiment, theendogenous non-human locus is a mouse locus, e.g., a mouse H-2K, H-2D orH-2L locus. In one embodiment, the non-human portion of the chimerichuman/non-human MHC I polypeptide comprises transmembrane andcytoplasmic domains of the endogenous non-human MHC I polypeptide. Thus,in an embodiment wherein the non-human animal is a mouse, the endogenousnon-human MHC I locus may be an H-2K locus (e.g., H-2Kb locus) and theendogenous non-human MHC I polypeptide may be an H-2K polypeptide;therefore, the chimeric human/non-human MHC I polypeptide may comprisetransmembrane and cytoplasmic domains of H-2K polypeptide. In anotherembodiment wherein the non-human animal is a mouse, the endogenousnon-human MHC I locus may be an H-2D locus and the endogenous non-humanMHC I polypeptide may be an H-2D polypeptide; therefore, the chimerichuman/non-human MHC I polypeptide may comprise transmembrane andcytoplasmic domains of H-2D polypeptide. Similarly, in anotherembodiment, the endogenous non-MHC I locus may be an H-2L locus and theendogenous non-human MHC I polypeptide may be an H-2L polypeptide;therefore, the chimeric human/non-human MHC I polypeptide may comprisetransmembrane and cytoplasmic domains of H-2L polypeptide.

Also provided herein is a mouse comprising at an endogenous H-2K locus anucleotide sequence encoding a chimeric human/mouse MHC I polypeptide,wherein a human portion of the chimeric polypeptide comprises anextracellular domain of a human HLA-A (e.g., HLA-A2) polypeptide and amouse portion comprises transmembrane and cytoplasmic domains of a mouseH-2K polypeptide, and wherein the mouse expresses the chimerichuman/mouse MHC I polypeptide. In some embodiments, the mouse does notexpress an extracellular domain of the mouse H-2K polypeptide from anendogenous H-2K locus. In one aspect, the nucleotide sequence encoding achimeric human/mouse MHC I polypeptide is operably linked to endogenousmouse regulatory elements. The human portion of the chimeric polypeptidemay comprise a human leader sequence. It may also comprise α1, α2, andα3 domains of the human MHC I polypeptide. The human MHC I polypeptidemay be HLA-A polypeptide, e.g., HLA-A2.1 polypeptide. In one aspect, themouse H-2K locus is an H-2Kb locus.

Another aspect of the invention relates to a non-human animal, e.g., arodent (e.g., a mouse or a rat), comprising in its genome a nucleotidesequence encoding a human or humanized β2 microglobulin polypeptide.Thus, provided herein is a non-human animal comprising at an endogenousnon-human β2 microglobulin locus a nucleotide sequence encoding a humanor humanized β2 microglobulin polypeptide, wherein the animal expressesthe human or humanized β2 microglobulin polypeptide. In one aspect, theanimal does not express a functional endogenous non-human β2microglobulin polypeptide from an endogenous non-human β2 microglobulinlocus. In one aspect, the animal comprises two copies of the β2microglobulin locus encoding the human or humanized β2 microglobulinpolypeptide; in another embodiment, the animal comprises one copy of theβ2 microglobulin locus encoding the human or humanized β2 microglobulinpolypeptide. Thus, the animal may be homozygous or heterozygous for theβ2 microglobulin locus encoding the human or humanized α2 microglobulinpolypeptide. In various embodiments, the nucleotide sequence encodingthe human or humanized β2 microglobulin polypeptide is comprised in thegermline of the non-human animal (e.g., rodent, e.g., rat or mouse). Inone embodiment, a nucleotide sequence encoding a human or humanized β2microglobulin polypeptide comprises a nucleotide sequence encoding apolypeptide comprising a human β2 microglobulin amino acid sequence. Inone embodiment, the polypeptide is capable of binding to an MHC Iprotein,

In some embodiments, the nucleotide sequence encoding the human orhumanized β2 microglobulin polypeptide is operably linked to endogenousnon-human β2 microglobulin regulatory elements. In one aspect, thenucleotide sequence encoding the human or humanized β2 microglobulinpolypeptide comprises a nucleotide sequence set forth in exon 2 to exon4 of a human β2 microglobulin gene. In another aspect, the nucleotidesequence encoding the human or humanized β2 microglobulin polypeptidecomprises nucleotide sequences set forth in exons 2, 3, and 4 of a humanβ2 microglobulin gene. In a further aspect, the nucleotide sequence alsocomprises a nucleotide sequence set forth in exon 1 of a non-human β2microglobulin gene. In some embodiments, the non-human animal is arodent (e.g., mouse or a rat); thus, the non-human β2 microglobulinlocus is a rodent (e.g., a mouse or a rat) β2 microglobulin locus.

Also provided is a mouse comprising at an endogenous β2 microglobulinlocus a nucleotide sequence encoding a human or humanized β2microglobulin polypeptide, wherein the mouse expresses the human orhumanized β2 microglobulin polypeptide. In some embodiments, the mousedoes not express a functional endogenous mouse β2 microglobulin from anendogenous β2 microglobulin locus. The nucleotide sequence may be linkedto endogenous mouse regulatory elements. In one aspect, the nucleotidesequence comprises a nucleotide sequence set forth in exon 2 to exon 4of a human β2 microglobulin gene. Alternatively, the nucleotide sequenceencoding the human or humanized β2 microglobulin polypeptide maycomprise nucleotide sequences set forth in exons 2, 3, and 4 of a humanβ2 microglobulin gene. The nucleotide sequence encoding the human orhumanized β2 microglobulin polypeptide may further comprise a nucleotidesequence of exon 1 of a mouse β2 microglobulin gene. In one embodiment,a nucleotide sequence encoding a human or humanized β2 microglobulinpolypeptide comprises a nucleotide sequence encoding a polypeptidecomprising a human β2 microglobulin amino acid sequence. In oneembodiment, the polypeptide is capable of binding to an MHC I protein.

The invention further provides a non-human animal (e.g., a rodent, e.g.,a mouse or a rat) comprising in its genome a nucleotide sequenceencoding a chimeric human/non-human MHC I polypeptide and a nucleotidesequence encoding a human or humanized β2 microglobulin polypeptide. Inone embodiment, the invention provides a non-human animal comprising inits genome a first nucleotide sequence encoding a chimerichuman/non-human MHC I polypeptide, wherein a human portion of thechimeric polypeptide comprises an extracellular domain of a human MHC Ipolypeptide; and a second nucleotide sequence encoding a human orhumanized β2 microglobulin polypeptide, wherein the first nucleotidesequence is located at an endogenous non-human MHC I locus, and thesecond nucleotide sequence is located at an endogenous non-human β2microglobulin locus, and wherein the animal expresses the chimerichuman/non-human MHC I polypeptide and the human or humanized β2microglobulin polypeptide. In one aspect, the animal is a mouse. Thus,the endogenous MHC I locus may be selected from a group consisting ofH-2K, H-2D, and H-2L locus. In one embodiment, the endogenous mouselocus is an H-2K locus (e.g., H-2Kb locus), In one embodiment, the humanMHC I polypeptide is selected from the group consisting of HLA-A, HLA-B,and HLA-C polypeptide. In one aspect, the human MHC I polypeptide isHLA-A, e.g., HLA-A2 (e.g., HLA-A2.1). In various embodiments, the firstand the second nucleotide sequences are comprised in the germline of thenon-human animal (e.g., rodent, e.g., mouse or rat).

Therefore, the invention provides a mouse comprising in its genome afirst nucleotide sequence encoding a chimeric human/mouse MHC Ipolypeptide, wherein a human portion of the chimeric polypeptidecomprises an extracellular domain of a human HLA-A (e.g., HLA-A2) and amouse portion comprises transmembrane and cytoplasmic domains of a mouseH-2K; and a second nucleotide sequence encoding a human or humanized β2microglobulin polypeptide, wherein the first nucleotide sequence islocated at an endogenous H-2K locus and the second nucleotide sequenceis located at an endogenous mouse β2 microglobulin locus, and whereinthe mouse expresses the chimeric human/mouse MHC I polypeptide and thehuman or humanized β2 microglobulin polypeptide. In one embodiment, thenon-human animal (e.g., the mouse) comprising both the chimeric MHC Ipolypeptide and human or humanized β2 microglobulin polypeptide does notexpress an extracellular domain of an endogenous non-human MHC Ipolypeptide (e.g., the mouse H-2K polypeptide) and/or a functionalendogenous non-human (e.g., the mouse) β2 microglobulin polypeptidesfrom their respective endogenous loci. In one aspect, the animal (e.g.,the mouse) comprises two copies of each of the first and the secondnucleotide sequence. In another aspect, the animal (e.g., the mouse)comprises one copy of the first and one copy of the second nucleotidesequences. Thus, the animal may be homozygous or heterozygous for boththe first and the second nucleotide sequences.

In one aspect, the first nucleotide sequence is operably linked toendogenous non-human (e.g., mouse) MHC I regulatory elements, and thesecond nucleotide sequence is operably linked to endogenous non-human(e.g., mouse) β2 microglobulin elements. The human portion of thechimeric polypeptide may comprise α1, α2, and α3 domains of the humanMHC I polypeptide. The second nucleotide sequence may comprise anucleotide sequence set forth in exon 2 to exon 4 of a human β2microglobulin gene. Alternatively, the second nucleotide sequence maycomprise nucleotide sequences set forth in exons 2, 3, and 4 of a humanβ2 microglobulin gene. In one aspect, the mouse comprising both thechimeric MHC I polypeptide and human or humanized β2 microglobulinpolypeptide may be such that the expression of human or humanized β2microglobulin increases the expression of the chimeric human/mouse MHC Ipolypeptide as compared to the expression of the chimeric human/mouseMHC I polypeptide in the absence of expression of human or humanized β2microglobulin polypeptide.

Also provided are methods of making genetically engineered non-humananimals (e.g., rodents, e.g., mice or rats) described herein. Thus, inone embodiment, provided is a method of modifying an MHC I locus of arodent (e.g., a mouse or a rat) to express a chimeric human/rodent(e.g., human/mouse or human/rat) MHC I polypeptide, wherein the methodcomprises replacing at the endogenous MHC I locus a nucleotide sequenceencoding an extracellular domain of a rodent MHC I polypeptide with anucleotide sequence encoding an extracellular domain of a human MHC Ipolypeptide. In another embodiment, provided is a method of modifying aβ2 microglobulin locus of a rodent (e.g., a mouse or a rat) to express ahuman or humanized β2 microglobulin polypeptide, wherein the methodcomprises replacing at the endogenous rodent (e.g., mouse or rat) β2microglobulin locus a nucleotide sequence encoding a rodent (e.g., amouse or a rat) β2 microglobulin polypeptide with a nucleotide sequenceencoding a human or humanized β2 microglobulin polypeptide. In suchmethods, the replacement may be made in a single ES cell, and the singleES cell may be introduced into a rodent (e.g., a mouse or a rat) to makean embryo. The resultant rodent (e.g., a mouse or a rat) can be bred togenerate a double humanized animal.

Thus, the invention also provides a method of making double humanizedanimals, e.g., rodents (e.g., mice or rats). In one embodiment, providedis a method of making a genetically modified mouse comprising (a)modifying an MHC I locus of a first mouse to express a chimerichuman/mouse MHC I polypeptide comprising replacing at the endogenousmouse MHC I locus a nucleotide sequence encoding an extracellular domainof a mouse MHC I polypeptide with a nucleotide sequence encoding anextracellular domain of a human MHC I polypeptide, (b) modifying a β2microglobulin locus of a second mouse to express a human or humanized β2microglobulin polypeptide comprising replacing at the endogenous mouseβ2 microglobulin locus a nucleotide sequence encoding a mouse β2microglobulin polypeptide with a nucleotide sequence encoding a human orhumanized β2 microglobulin polypeptide; and (c) breeding the first andthe second mouse to generate a genetically modified mouse comprising inits genome a first nucleotide sequence encoding a chimeric human/mouseMHC I polypeptide and a second nucleotide sequence encoding a human orhumanized β2 microglobulin polypeptide, wherein the genetically modifiedmouse expresses the chimeric human/mouse MHC I polypeptide and the humanor humanized β2 microglobulin polypeptide. In some embodiments, the MHCI locus is selected from H-2K, H-2D, and H-2L; in some embodiments, thehuman MHC I polypeptide is selected from HLA-A, HLA-B, and HLA-C. In oneembodiment, the MHC I locus is an H-2K locus, the human MHC Ipolypeptide is HLA-A (e.g., HLA-A2), and the mouse expresses a chimericHLA-A/H-2K polypeptide (e.g., HLA-A2/H-2K polypeptide). In one aspect,the chimeric HLA-A2/H-2K polypeptide comprises an extracellular domainof the HLA-A2 polypeptide and cytoplasmic and transmembrane domains ofH-2K polypeptide. In one aspect, the second nucleotide sequencecomprises nucleotide sequences set forth in exons 2, 3, and 4 (e.g.,exon 2 to exon 4) of a human β2 microglobulin gene, and a nucleotidesequence set forth in exon 1 of a mouse β2 microglobulin gene.

Also provided herein are cells, e.g., isolated antigen-presenting cells,derived from the non-human animals (e.g., rodents, e.g., mice or rats)described herein. Tissues and embryos derived from the non-human animalsdescribed herein are also provided.

In yet another embodiment, the invention provides methods foridentification of antigens or antigen epitopes that elicit immuneresponse, methods for evaluating a vaccine candidate, methods foridentification of high affinity T cells to human pathogens or cancerantigens.

Any of the embodiments and aspects described herein can be used inconjunction with one another, unless otherwise indicated or apparentfrom the context. Other embodiments will become apparent to thoseskilled in the art from a review of the ensuing detailed description.The following detailed description includes exemplary representations ofvarious embodiments of the invention, which are not restrictive of theinvention as claimed. The accompanying figures constitute a part of thisspecification and, together with the description, serve only toillustrate embodiments and not to limit the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the four domains of a class I MHCmolecule: α-chain containing the α1, α2 and α3 domains and thenon-covalently associated fourth domain, β2 -microglobulin Wm). The graycircle represents a peptide bound in the peptide-binding cleft.

FIG. 2 is a schematic representation (not to scale) of the relativegenomic structure of the human HLA, showing class I, II and III genes.

FIG. 3 is a schematic representation (not to scale) of the relativegenomic structure of the mouse MHC, showing class I, II and III genes.

FIG. 4 illustrates a viral vector construct containing a cDNA encoding achimeric HLA-A/H-2K polypeptide with an IRES-GFP reporter (A); andhistograms comparing expression of human HLA-A2 in MG87 cells transducedwith HLA-A2 (dashed line), HLA-A2/H-2K (dotted line), or no transduction(solid line) either alone (left) or co-transduced with humanized β2microglobulin (right) (B). Data from horizontal gates presentedgraphically in (B) is illustrated as percent of cells expressing theconstruct in the table in (C).

FIG. 5 is a schematic diagram (not to scale) of the targeting strategyused for making a chimeric H-2K locus that expresses an extracellularregion of a human HLA-A2 protein. Mouse sequences are represented inblack and human sequences are represented in white. La=leader,UTR=untranslated region, TM=transmembrane domain, CYT=cytoplasmicdomain, HYG=hygromycin.

FIG. 6A demonstrates expression (% total cells) of HLA-A2 (left) andH-2K (right) in cells isolated from either a wild-type (WT) mouse or aheterozygous mouse carrying the chimeric HLA-A2/H-2K locus (HLA-A/H-2KHET).

FIG. 6B is a dot plot of in vivo expression of the chimeric HLA-A2/H-2Kprotein in a heterozygous mouse harboring a chimeric HLA-A2/H-2K locus.

FIG. 7 shows a targeting strategy (not to scale) for humanization of aβ2 microglobulin gene at a mouse β2 microglobulin locus. Mouse sequencesare in black and human sequences are in white. NEO=neomycin.

FIG. 8 shows a representative dot plot of HLA class I and human β2microglobulin expression on cells isolated from the blood of wild-type(WT) mice, mice heterozygous for chimeric HLA-A2/H-2K, and miceheterozygous for chimeric HLA-A2/H-2K and heterozygous for humanized β2microglobulin (double heterozygous; class I/β2m HET).

FIG. 9 shows a representative histogram of human HLA class I expression(X axis) on cells isolated from the blood of wild-type (WT), chimericHLA-A2/H-2K heterozygous (class I HET), and chimericHLA-A2/H2K/humanized β2 microglobulin double heterozygous (class I/β2mHET) mice.

FIG. 10 shows the results of IFNγ Elispot assays for human T cellsexposed to antigen-presenting cells (APCs) from wild-type mice (WT APCs)or mice heterozygous for both chimeric HLA-A2/H-2K and humanized β2microglobulin (double HET APCs) in the presence of flu (left) or EBV(right) peptides. Statistical analysis was performed using one way ANOVAwith a Tukey's Multiple Comparison Post Test.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The present invention provides genetically modified non-human animals(e.g., mice, rats, rabbits, etc.) that express human or humanized MHC Iand/or β2 microglobulin polypeptides; embryos, cells, and tissuescomprising the same; methods of making the same; as well as methods ofusing the same. Unless defined otherwise, all terms and phrases usedherein include the meanings that the terms and phrases have attained inthe art, unless the contrary is clearly indicated or clearly apparentfrom the context in which the term or phrase is used.

The term “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). Conservative amino acidsubstitutions may be achieved by modifying a nucleotide sequence so asto introduce a nucleotide change that will encode the conservativesubstitution. In general, a conservative amino acid substitution willnot substantially change the functional properties of interest of aprotein, for example, the ability of MHC I to present a peptide ofinterest. Examples of groups of amino acids that have side chains withsimilar chemical properties include aliphatic side chains such asglycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxylside chains such as serine and threonine; amide-containing side chainssuch as asparagine and glutamine; aromatic side chains such asphenylalanine, tyrosine, and tryptophan; basic side chains such aslysine, arginine, and histidine; acidic side chains such as asparticacid and glutamic acid; and, sulfur-containing side chains such ascysteine and methionine. Conservative amino acids substitution groupsinclude, for example, valine/leucine/isoleucine, phenylalanine/tyrosine,lysine/arginine, alanine/valine, glutamate/aspartate, andasparagine/glutamine. In some embodiments, a conservative amino acidsubstitution can be a substitution of any native residue in a proteinwith alanine, as used in, for example, alanine scanning mutagenesis. Insome embodiments, a conservative substitution is made that has apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. ((1992) Exhaustive Matching of the Entire Protein SequenceDatabase, Science 256:1443-45), hereby incorporated by reference. Insome embodiments, the substitution is a moderately conservativesubstitution wherein the substitution has a nonnegative value in thePAM250 log-likelihood matrix.

Thus, also encompassed by the invention is a genetically modifiednon-human animal whose genome comprises a nucleotide sequence encoding ahuman or humanized MHC I polypeptide and/or β2 microglobulinpolypeptide, wherein the polypeptide(s) comprises conservative aminoacid substitutions of the amino acid sequence(s) described herein.

One skilled in the art would understand that in addition to the nucleicacid residues encoding a human or humanized MHC I polypeptide and/or β2microglobulin described herein, due to the degeneracy of the geneticcode, other nucleic acids may encode the polypeptide(s) of theinvention. Therefore, in addition to a genetically modified non-humananimal that comprises in its genome a nucleotide sequence encoding MHC Iand/or β2 microglobulin polypeptide(s) with conservative amino acidsubstitutions, a non-human animal whose genome comprises a nucleotidesequence(s) that differs from that described herein due to thedegeneracy of the genetic code is also provided.

The term “identity” when used in connection with sequence includesidentity as determined by a number of different algorithms known in theart that can be used to measure nucleotide and/or amino acid sequenceidentity. In some embodiments described herein, identities aredetermined using a ClustalW v. 1.83 (slow) alignment employing an opengap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnetsimilarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The lengthof the sequences compared with respect to identity of sequences willdepend upon the particular sequences. In various embodiments, identityis determined by comparing the sequence of a mature protein from itsN-terminal to its C-terminal. In various embodiments when comparing achimeric human/non-human sequence to a human sequence, the human portionof the chimeric human/non-human sequence (but not the non-human portion)is used in making a comparison for the purpose of ascertaining a levelof identity between a human sequence and a human portion of a chimerichuman/non-human sequence (e.g., comparing a human ectodomain of achimeric human/mouse protein to a human ectodomain of a human protein).

The terms “homology” or “homologous” in reference to sequences, e.g.,nucleotide or amino acid sequences, means two sequences which, uponoptimal alignment and comparison, are identical in at least about 75% ofnucleotides or amino acids, at least about 80% of nucleotides or aminoacids, at least about 90-95% nucleotides or amino acids, e.g., greaterthan 97% nucleotides or amino acids. One skilled in the art wouldunderstand that, for optimal gene targeting, the targeting constructshould contain arms homologous to endogenous DNA sequences (i.e.,“homology arms”); thus, homologous recombination can occur between thetargeting construct and the targeted endogenous sequence.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. As such, a nucleic acid sequenceencoding a protein may be operably linked to regulatory sequences (e.g.,promoter, enhancer, silencer sequence, etc.) so as to retain propertranscriptional regulation. In addition, various portions of thechimeric or humanized protein of the invention may be operably linked toretain proper folding, processing, targeting, expression, and otherfunctional properties of the protein in the cell. Unless statedotherwise, various domains of the chimeric or humanized proteins of theinvention are operably linked to each other.

The term “MHC I complex” or the like, as used herein, includes thecomplex between the MHC I α chain polypeptide and the β2-microglobulinpolypeptide. The term “MHC I polypeptide” or the like, as used herein,includes the MHC I α chain polypeptide alone. Typically, the terms“human MHC” and “HLA” can be used interchangeably.

The term “replacement” in reference to gene replacement refers toplacing exogenous genetic material at an endogenous genetic locus,thereby replacing all or a portion of the endogenous gene with anorthologous or homologous nucleic acid sequence. As demonstrated in theExamples below, nucleic acid sequences of endogenous loci encodingportions of mouse MHC I and β2 microglobulin polypeptides were replacedby nucleotide sequences encoding portions of human MHC I and β2microglobulin polypeptides, respectively.

“Functional” as used herein, e,g., in reference to a functionalpolypeptide, refers to a polypeptide that retains at least onebiological activity normally associated with the native protein. Forexample, in some embodiments of the invention, a replacement at anendogenous locus (e.g., replacement at an endogenous non-human MHC Iand/or β2 microglobulin locus) results in a locus that fails to expressa functional endogenous polypeptide.

Several aspects described herein below for the genetically modified MHCI non-human animals, e.g., animal type; animal strains; cell types;screening, detection and other methods: methods of use; etc., will beapplicable to the genetically engineered β2 microglobulin and MHC I/β2microglobulin animals.

Genetically Modified MHC I Animals

In various embodiments, the invention generally provides geneticallymodified non-human animals that comprise in their genome a nucleotidesequence encoding a human or humanized MHC I polypeptide; thus, theanimals express a human or humanized MHC I polypeptide.

MHC genes are categorized into three classes: class I, class II, andclass III, all of which are encoded either on human chromosome 6 ormouse chromosome 17, A schematic of the relative organization of thehuman and mouse MHC classes is presented in FIGS. 2 and 3, respectively.The MHC genes are among the most polymorphic genes of the mouse andhuman genomes. MHC polymorphisms are presumed to be important inproviding evolutionary advantage; changes in sequence can result indifferences in peptide binding that allow for better presentation ofpathogens to cytotoxic T cells.

MHC class I protein comprises an extracellular domain (which comprisesthree domains: α₁, α₂, and α₃), a transmembrane domain, and acytoplasmic tail. The α₁ and α₂ domains form the peptide-binding cleft,while the α₃ interacts with β2-microglobulin.

In addition to its interaction with β2-microglobulin, the α₃ domaininteracts with the TCR co-receptor CD8, facilitating antigen-specificactivation. Although binding of MHC class I to CD8 is about 100-foldweaker than binding of TCR to MHC class I, CD8 binding enhances theaffinity of TCR binding. Wooldridge et al. (2010) MHC Class I Moleculeswith Superenhanced CD8 Binding Properties Bypass the Requirement forCognate TCR Recognition and Nonspecifically Activate CTLs, J. Immuno!,184:3357-3366. Interestingly, increasing MHC class I binding to CD8abrogated antigen specificity in CTL activation. Id.

CD8 binding to MHC class I molecules is species-specific; the mousehomolog of CD8, Lyt-2, was shown to bind H-2D^(d) molecules at the α3domain, but it did not bind HLA-A molecules. Connolly et al. (1988) TheLyt-2 Molecule Recognizes Residues in the Class I α3 Domain inAllogeneic Cytotoxic T Cell Responses, J. Exp. Med. 168:325-341.Differential binding was presumably due to CDR-like determinants (CDR1-and CDR2-like) on CD8 that was not conserved between humans and mice.Sanders et al. (1991) Mutations in CD8 that Affect Interactions with HLAClass I and Monoclonal Anti-CD8 Antibodies, J. Exp. Med. 174:371-379;Vitiello et al. (1991) Analysis of the HLA-restricted Influenza-specificCytotoxic T Lymphocyte Response in Transgenic Mice Carrying a ChimericHuman-Mouse Class I Major Histocompatibility Complex, J. Exp. Med.173:1007-1015; and, Cao et al. (1997) Crystal structure of the complexbetween human CD8αα and HLA-A2, Nature 387:630-634. It has been reportedthat CD8 binds HLA-A2 in a conserved region of the α3 domain (atposition 223-229). A single substitution (V245A) in HLA-A reducedbinding of CD8 to HLA-A, with a concomitant large reduction in Tcell-mediated lysis. Salter et al. (1989), Polymorphism in the α₃ domainof HLA-A molecules affects binding to CD8, Nature 338:345-348. Ingeneral, polymorphism in the α3 domain of HLA-A molecules also affectedbinding to CD8. Id. In mice, amino acid substitution at residue 227 inH-2D′′ affected the binding of mouse Lyt-2 to H-2D^(d), and cellstransfected with a mutant H-2D^(d) were not lysed by CD8+ T cells.Potter et al. (1989) Substitution at residue 227 of H-2 class Imolecules abrogates recognition by CD8-dependent, but notCD8-independent, cytotoxic T lymphocytes, Nature 337:73-75.

Therefore, due to species specificity of interaction between the MHCclass I α3 domain and CD8, an MHC I complex comprising a replacement ofan H-2K α3 domain with a human HLA-A2 α3 domain was nonfunctional in amouse (i.e., in vivo) in the absence of a human CD8. In animalstransgenic for HLA-A2, substitution of human α3 domain for the mouse α3domain resulted in restoration of T cell response. Irwin et al. (1989)Species-restricted interactions between CD8 and the α3 domain of class Iinfluence the magnitude of the xenogeneic response, J. Exp. Med.170:1091-1101; Vitiello et al. (1991), supra.

The transmembrane domain and cytoplasmic tail of mouse MHC class Iproteins also have important functions. One function of MHC Itransmembrane domain is to facilitate modulation by HLA-A2 of homotypiccell adhesion (to enhance or inhibit adhesion), presumably as the resultof cross-linking (or ligation) of surface MHC molecules. Wagner et al.(1994) Ligation of MHC Class I and Class II Molecules Can Lead toHeterologous Desensitization of Signal Transduction Pathways ThatRegulate Homotypic Adhesion in Human Lymphocytes, J. Immunol.152:5275-5287. Cell adhesion can be affected by mAbs that bind atdiverse epitopes of the HLA-A2 molecule, suggesting that there aremultiple sites on HLA-A2 implicated in modulating homotypic celladhesion; depending on the epitope bound, the affect can be to enhanceor to inhibit HLA-A2-dependent adhesion. Id.

The cytoplasmic tail, encoded by exons 6 and 7 of the MHC I gene, isreportedly necessary for proper expression on the cell surface and forLIR1-mediated inhibition of NK cell cytotoxicity. Gruda et al. (2007)Intracellular Cysteine Residues in the Tall of MHC Class I Proteins AreCrucial for Extracellular Recognition by Leukocyte Ig-Like Receptor 1,J. Immunol. 179:3655-3661. A cytoplasmic tail is required formultimerizaton of at least some MHC I molecules through formation ofdisulfide bonds on its cysteine residues, and thus may play a role inclustering and in recognition by NK cells. Lynch et al. (2009) Novel MHCClass I Structures on Exosomes, J. Immunol, 183:1884-1891.

The cytoplasmic domain of HLA-A2 contains a constitutivelyphosphorylated serine residue and a phosphorylatable tyrosine,although—in Jurkat cells—mutant HLA-A2 molecules lacking a cytoplasmicdomain appear normal with respect to expression, cytoskeletalassociation, aggregation, and endocytic internalization. Gur et al.(1997) Structural Analysis of Class I MHC Molecules: The CytoplasmicDomain Is Not Required for Cytoskeletal Association, Aggregation, andInternalization, Mol. Immunol. 34(2):125-132. Truncated HLA-A2 moleculeslacking the cytoplasmic domain are apparently normally expressed andassociate with β2 microglobulin. Id.

However, several studies have demonstrated that the cytoplasmic tail iscritical in intracellular trafficking, dendritic cell (DC)-mediatedantigen presentation, and CTL priming. A tyrosine residue encoded byexon 6 was shown to be required for MHC I trafficking through endosomalcompartments, presentation of exogenous antigens, and CTL priming; whiledeletion of exon 7 caused enhancement of anti-viral CTL responses. Lizeeet al. (2003) Control of Dendritic Cross-Presentation by the MajorHistocompatibility Complex Class I Cytoplasmic Domain, Nature Immunol,4:1065-73; Basha et al. (2008) MHC Class I Endosomal and LysosomalTrafficking Coincides with Exogenous Antigen Loading in Dendritic Cells,PLoS ONE 3: e3247; and Rodriguez-Cruz et al. (2011) Natural SpliceVariant of MHC Class I Cytoplasmic Tail Enhances Dendritic Cell-InducedCD8+ T-Cell Responses and Boosts Anti-Tumor Immunity, PLoS ONE 6:e22939.

In various embodiments, the invention provides a genetically modifiednon-human animal (e.g., mouse, rat, rabbit, etc.) that comprises in itsgenome a nucleotide sequence encoding a human or humanized MHC class Ipolypeptide. The non-human animal may comprise in its genome anucleotide sequence that encodes an MHC I polypeptide that is partiallyhuman and partially non-human, e.g., a non-human animal that expresses achimeric human/non-human MHC I polypeptide. In one aspect, the non-humananimal only expresses the human or humanized MHC I polypeptide, e.g.,chimeric human/non-human MHC I polypeptide, and does not express anendogenous non-human MHC I protein from an endogenous MHC I locus.

In one embodiment, the chimeric human/non-human MHC I polypeptidecomprises in its human portion a peptide binding domain of a human MHC Ipolypeptide. In one aspect, the human portion of the chimericpolypeptide comprises an extracellular domain of a human MHC I. In thisembodiment, the human portion of the chimeric polypeptide comprises anextracellular domain of an a chain of a human MHC I. In one embodiment,the human portion of the chimeric polypeptide comprises α1 and α2domains of a human MHC I. In another embodiment, the human portion ofthe chimeric polypeptide comprises α1, α2, and α3 domains of a human MHCI.

The human or humanized MHC I polypeptide may be derived from afunctional human HLA molecule encoded by any of HLA-A, HLA-B, HLA-C,HLA-E, HLA-F, or HLA-G loci. A list of commonly used HLA antigens isdescribed in Shankarkumar et al. ((2004) The Human Leukocyte Antigen(HLA) System, Int. Hum. Genet. 4(2):91-103), incorporated herein byreference. Shankarkumar et al. also present a brief explanation of HLAnomenclature used in the art Additional information regarding HLAnomenclature and various HLA alleles can be found in Holdsworth et al.(2009) The HLA dictionary 2008: a summary of HLA-A, -B, -C, -DRB1/3/4/5,and DQB1 alleles and their association with serologically defined HLA-A,-B, -C, -DR, and -DQ antigens, Tissue Antigens 73:95-170, and a recentupdate by Marsh et al. (2010) Nomenclature for factors of the HLAsystem, 2010, Tissue Antigens 75:291-455, both incorporated herein byreference. Thus, the human or humanized MHC I polypeptide may be derivedfrom any functional human HLA class I molecules described therein.

In one specific aspect, the human or humanized MHC I polypeptide isderived from human HLA-A. In a specific embodiment, the HLA-Apolypeptide is an HLA-A2 polypeptide (e.g., and HLA-A2.1 polypeptide).In one embodiment, the HLA-A polypeptide is a polypeptide encoded by anHLA-A*0201 allele, e.g., HLA-A*02:01:01:01 allele. The HLA-A*0201 alleleis commonly used amongst the North American population. Although thepresent Examples describe this particular HLA sequence, any suitableHLA-A sequence is encompassed herein, e.g., polymorphic variants ofHLA-A2 exhibited in human population, sequences with one or moreconservative or non-conservative amino acid modifications, nucleic acidsequences differing from the sequence described herein due to thedegeneracy of genetic code, etc.

In one aspect, a non-human animal that expresses a human HLA-A2 sequenceis provided, wherein the human HLA-A2 sequence comprises one or moreconservative or non-conservative modifications.

In one aspect, a non-human animal that expresses a human HLA-A2 sequenceis provided, wherein the human HLA-A2 sequence is at least about 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to a human HLA-A2 sequence. Ina specific embodiment, the human HLA-A2 sequence is at least about 90%,95%, 96%, 97%, 98%, or 99% identical to the human HLA-A2 sequencedescribed in the Examples. In one embodiment, the human HLA-A2 sequencecomprises one or more conservative substitutions. In one embodiment, thehuman HLA-A2 sequence comprises one or more non-conservativesubstitutions.

In another specific aspect, the human or humanized MHC I polypeptide isderived from human MHC I selected from HLA-B and HLA-C. In one aspect,the human or humanized MHC I is derived from HLA-B, e.g., HLA-B27.

In one aspect, the non-human portion of the chimeric human/non-human MHCI polypeptide comprises transmembrane and/or cytoplasmic domains of thenon-human MHC I polypeptide. In one embodiment, the non-human animal isa mouse, and the non-human MHC I polypeptide is selected from H-2K,H-2D, and H-2L. In one embodiment, the non-human MHC I polypeptide isH-2K, e.g., H-2Kb. Although specific H-2K sequences are described in theExamples, any suitable H-2K sequences, e.g., polymorphic variants,conservative/non-conservative amino acid substitutions, etc., areencompassed herein.

The non-human animal described herein may comprise in its genome anucleotide sequence encoding a human or humanized MHC I polypeptide,e.g., chimeric human/non-human MHC I polypeptide, wherein the nucleotidesequence encoding such polypeptide is located at an endogenous non-humanMHC I locus (e.g., H-2K locus). In one aspect, this results in areplacement of an endogenous MHC I gene or a portion thereof with anucleotide sequence encoding a human or humanized MHC I polypeptide,e.g., a chimeric gene encoding a chimeric human/non-human MHC Ipolypeptide described herein. In one embodiment, the replacementcomprises a replacement of an endogenous nucleotide sequence encoding anon-human MHC I peptide binding domain or a non-human MHC Iextracellular domain with a human nucleotide sequence (e,g., HLA-A2nucleotide sequence) encoding the same. In this embodiment, thereplacement does not comprise a replacement of an MHC I sequenceencoding transmembrane and/or cytoplasmic domains of a non-human MHC Ipolypeptide (e.g., H-2K polypeptide). Thus, the non-human animalcontains chimeric human/non-human nucleotide sequence at an endogenousnon-human MHC I locus, and expresses chimeric human/non-human MHCpolypeptide from the endogenous non-human MHC I locus.

A chimeric human/non-human polypeptide may be such that it comprises ahuman or a non-human leader (signal) sequence. In one embodiment, thechimeric polypeptide comprises a non-human leader sequence of anendogenous MHC I protein. In another embodiment, the chimericpolypeptide comprises a leader sequence of a human MHC I protein, e.g.,HLA-A2 protein (for instance, HLA-A2.1 leader sequence). Thus, thenucleotide sequence encoding the chimeric MHC I polypeptide may beoperably linked to a nucleotide sequence encoding a human MHC I leadersequence.

A chimeric human/non-human MHC I polypeptide may comprise in its humanportion a complete or substantially complete extracellular domain of ahuman MHC I polypeptide. Thus, the human portion may comprise at least80%, preferably at least 85%, more preferably at least 90%, e.g., 95% ormore of the amino acids encoding an extracellular domain of a human MHCI polypeptide (e.g., HLA-A2 polypeptide). In one example, substantiallycomplete extracellular domain of the human MHC I polypeptide lacks ahuman MHC I leader sequence. In another example, the chimerichuman/non-human MHC I polypeptide comprises a human MHC I leadersequence.

Moreover, the chimeric MHC I polypeptide may be expressed under thecontrol of endogenous non-human regulatory elements, e.g., rodent MHC Iregulatory animals. Such arrangement will facilitate proper expressionof the chimeric MHC I polypeptide in the non-human animal, e.g., duringimmune response in the non-human animal.

The genetically modified non-human animal may be selected from a groupconsisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull,buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g.,marmoset, rhesus monkey). For the non-human animals where suitablegenetically modifiable ES cells are not readily available, other methodsare employed to make a non-human animal comprising the geneticmodification. Such methods include, e.g., modifying a non-ES cell genome(e.g., a fibroblast or an induced pluripotent cell) and employingnuclear transfer to transfer the modified genome to a suitable cell,e.g., an oocyte, and gestating the modified cell (e.g., the modifiedoocyte) in a non-human animal under suitable conditions to form anembryo.

In one aspect, the non-human animal is a mammal. In one aspect, thenon-human animal is a small mammal, e.g., of the superfamily Dipodoideaor Muroidea. In one embodiment, the genetically modified animal is arodent. In one embodiment, the rodent is selected from a mouse, a rat,and a hamster. In one embodiment, the rodent is selected from thesuperfamily Muroidea. In one embodiment, the genetically modified animalis from a family selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae(climbing mice, rock mice, with-tailed rats, Malagasy rats and mice),Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., molerates, bamboo rats, and zokors). In a specific embodiment, thegenetically modified rodent is selected from a true mouse or rat (familyMuridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment,the genetically modified mouse is from a member of the family Muridae.In one embodiment, the animal is a rodent. In a specific embodiment, therodent is selected from a mouse and a rat. In one embodiment, thenon-human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent that is amouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa,C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, themouse is a 129 strain selected from the group consisting of a strainthat is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/Svlm),129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8,129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature forstrain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al(2000) Establishment and Chimera Analysis of 129/SvEv- andC57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specificembodiment, the genetically modified mouse is a mix of an aforementioned129 strain and an aforementioned C57BL/6 strain. In another specificembodiment, the mouse is a mix of aforementioned 129 strains, or a mixof aforementioned BL/6 strains. In a specific embodiment, the 129 strainof the mix is a 12986 (129/SvEvTac) strain. In another embodiment, themouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment,the mouse is a mix of a BALB strain and another aforementioned strain.

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

Thus, in one embodiment, the invention relates to a genetically modifiedmouse that comprises in its genome a nucleotide sequence encoding achimeric human/mouse MHC I polypeptide, wherein a human portion of thechimeric polypeptide comprises a peptide binding domain or anextracellular domain of a human MHC I (e.g., human HLA-A, e.g., humanHLA-A2, e.g., human HLA-A2.1). In some embodiments, the mouse does notexpress a peptide binding or an extracellular domain of an endogenousmouse polypeptide from its endogenous mouse locus. The peptide bindingdomain of the human MHC I may comprise α1 and α2 domains. Alternatively,the peptide binding domain of the human MHC I may comprise α1, α2, andα3 domains. In one aspect, the extracellular domain of the human MHC Icomprises an extracellular domain of a human MHC I α chain. In oneembodiment, the endogenous mouse locus is an H-2K (e.g., H-2Kb) locus,and the mouse portion of the chimeric polypeptide comprisestransmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb)polypeptide.

Thus, in one embodiment, a genetically modified mouse is provided,wherein the mouse comprises at an endogenous H-2K (e.g., H-2Kb) locus anucleotide sequence encoding a chimeric human/mouse MHC I polypeptide,wherein a human portion of the chimeric polypeptide comprises anextracellular domain of a human HLA-A2 (e.g., HLA-A2.1) polypeptide anda mouse portion comprises transmembrane and cytoplasmic domains of amouse H-2K (e.g., H-2Kb) polypeptide. In one aspect, the mouse does notexpress an extracellular domain of the mouse H-2K (e.g., H-2Kb)polypeptide from an endogenous MHC I locus. In one embodiment, the mouseexpresses a chimeric HLA-A2/H-2K (e.g., a chimeric HLA-A2.1/H-2Kb)polypeptide from an endogenous H-2K (e.g., H-2Kb) locus. In variousembodiments, expression of the chimeric gene is under control ofendogenous mouse MHC class I regulatory elements. In some aspects, themouse comprises two copies of the chimeric MHC I locus containing anucleotide sequence encoding a chimeric HLA-A2/H-2K polypeptide; whilein other aspects, the mouse comprises one copy of the chimeric MHC Ilocus containing a nucleotide sequence encoding a chimeric HLA-A2/H-2Kpolypeptide. Thus, the mouse may be homozygous or heterozygous for thenucleotide sequence encoding the chimeric HLA-A2/H-2K polypeptide.

In some embodiments described herein, a mouse is provided that comprisesa chimeric MHC I locus located at an endogenous mouse H-2K locus. Thechimeric locus comprises a nucleotide sequence that encodes anextracellular domain of a human HLA-A2 protein, e.g., αa1, α2, and α3domains of a human HLA-A2 gene. The chimeric locus lacks a nucleotidesequence that encodes an extracellular domain of a mouse H-2K protein(e.g., α1, α2, and α3 domains of the mouse H-2K). In one aspect, thechimeric locus lacks a nucleotide sequence that encodes a leaderpeptide, α1, α2, and α3 domains of a mouse H-2K; and comprises a leaderpeptide, α1, α2, and α3 domains of a human HLA-A2, and transmembrane andcytoplasmic domains of a mouse H-2K. The various domains of the chimericlocus are operably linked to each other such that the chimeric locusexpresses a functional chimeric human/mouse MHC I protein.

In various embodiments, a non-human animal, e.g., a rodent (e.g., amouse or a rat), that expresses a functional chimeric MHC I protein froma chimeric MHC I locus as described herein displays the chimeric proteinon a cell surface. In one embodiment, the non-human animal expressed thechimeric MHC I protein on a cell surface in a cellular distribution thatis the same as observed in a human. In one aspect, the cell displays apeptide fragment (an antigen fragment) bound to an extracellular portion(e.g., human HLA-A2 extracellular portion) of the chimeric MHC Iprotein. In an embodiment, the extracellular portion of such chimericprotein interacts with other proteins on the surface of said cell, e.g.,β2-microglobulin.

In various embodiments, a cell displaying the chimeric MHC I protein,e.g., HLA-A2/H-2K protein, is a nucleated cell. In various aspects, thecell is an antigen-presenting cell (APC). Although most cells in thebody can present an antigen in the context of MHC I. some nonlimitingexamples of antigen presenting cells include macrophages, dendriticcells, and B cells. Other antigen presenting cells, includingprofessional and nonprofessional APCs, are known in the art, and areencompassed herein. In some embodiments, the cell displaying thechimeric MHC I protein is a tumor cell, and a peptide fragment presentedby the chimeric protein is derived from a tumor. In other embodiments,the peptide fragment presented by the chimeric MHC I protein is derivedfrom a pathogen, e.g., a bacterium or a virus.

The chimeric MHC I protein described herein may interact with otherproteins on the surface of the same cell or a second cell. In someembodiments, the chimeric MHC I protein interacts with endogenousnon-human proteins on the surface of said cell. The chimeric MHC proteinmay also interact with human or humanized proteins on the surface of thesame cell or a second cell.

On the same cell, HLA class I molecules may functionally interact withboth non-human (e.g., rodent, e.g., mouse or rat) and human β2-microglobulin. Thus, in one embodiment, the chimeric MHC I protein,e.g., HLA-A2/H-2K protein, interacts with a mouse β2 -microglobulin.Although interaction between some human HLA class I molecules and mouseβ32-microglobulin is possible, it nevertheless may be greatly reduced incomparison to interaction between human HLA class I and humanβ2-microglobulin. Thus, in the absence of human β2-microglobulin,expression of human MHC I on the cell surface may be reduced. Perarnauet al. (1988) Human β2 -microglobulin Specifically Enhances Cell-SurfaceExpression of HLA Class I Molecules in Transfected Murine Cells, J.Immunol. 141:1383-89. Other HLA molecules, e.g., HLA-B27, do notinteract with mouse β2-microglobulin; see, e.g., Tishon et al. (2000)Transgenic Mice Expressing Human HLA and CD8 Molecules GenerateHLA-Restricted Measles Virus Cytotoxic T Lymphocytes of the SameSpecificity as Humans with Natural Measles Virus Infection, Virology275:286-293, which reports that HLA-827 function in transgenic micerequires both human β2-microglobulin and human CD8. Therefore, inanother embodiment, the chimeric MHC I protein interacts with a human orhumanized β2-microglobulin. In some such embodiments, as describedherein below, the non-human animal, e,g, a rodent (e.g., a mouse or arat), comprises in its genome a human or humanized β2-microglobulingene, and the animal expresses a functional human or humanizedβ2-microglobulin polypeptide; therefore, the chimeric MHC I proteininteracts with a human or humanized β2-microglobulin polypeptide.

In various aspects, the chimeric protein (e.g., HLA-A2/H-2K protein)also interacts with proteins on the surface of a second cell (throughits extracellular portion). The second cell may be a cell of anon-human, e.g., a mouse, or a human origin. The second cell may bederived from the same non-human animal or the same non-human animalspecie as the cell expressing the chimeric MHC I polypeptide.Nonlimiting examples of proteins with which the extracellular portion ofa chimeric protein (e.g., HLA-A2/H-2K) may interact include T cellreceptor (TCR) and its co-receptor CD8. Thus, a second cell may be a Tcell. In addition, the extracellular portion of the chimeric MHC Iprotein may bind a protein on the surface of Natural Killer (NK) cells,e.g., killer immunoglobulin receptors (KIRs) on the surface of NK cells.

A T cell or NK cell may bind a complex formed between the chimeric MHC Ipolypeptide and its displayed peptide fragment. Such binding may resultin T cell activation or inhibition of NK-mediated cell killing,respectively. One hypothesis is that NK cells have evolved to killeither infected or tumor cells that have evaded T cell mediatedcytotoxicity by downregulating their MHC I complex. However, when MHC Icomplex is expressed on cell surface, NK cell receptors recognize it,and NK-mediated cell killing is inhibited. Thus, in some aspects, whenan NK cell binds a complex formed between the chimeric MHC I polypeptide(e.g., HLA-A2/H-2K polypeptide) and a displayed peptide fragment on thesurface of infected or tumor cell, the NK-mediated cell killing isinhibited.

In one example, the chimeric MHC I polypeptide described herein, e.g., achimeric HLA-A2/H-2K polypeptide, interacts with CD8 protein on thesurface of a second cell. In one embodiment, the chimeric HLA-A2/H-2Kpolypeptide interacts with endogenous rodent (e.g., mouse or rat) CD8protein on the surface of a second cell. In one embodiment, the secondcell is a T cell. In another embodiment, the second cell is engineeredto express CD8. In certain aspects, the chimeric HLA-A2/H-2K polypeptideinteracts with a human CD8 on the surface of the second cell (e.g., ahuman cell or a rodent cell). In some such embodiments, the non-humananimal, e.g., a mouse or a rat, comprises a human CD8 transgene, and themouse or the rat expresses a functional human CD8 protein.

The chimeric MHC I polypeptide described herein may also interact with anon-human (e.g., a mouse or a rat) TCR, a human TCR, or a humanized TCRon a second cell. The chimeric MHC I polypeptide may interact with anendogenous TCR (e.g., mouse or rat TCR) on the surface of a second cell.The chimeric MHC I polypeptide may also interact with a human orhumanized TCR expressed on the surface of a second cell, wherein thecell is derived from the same animal or animal specie (e.g., mouse orrat) as the cell expressing the chimeric MHC I polypeptide. The chimericMHC I polypeptide may interact with a human TCR expressed on the surfaceof a human cell.

In addition to genetically engineered non-human animals, a non-humanembryo (e.g., a rodent embryo, e.g., mouse or a rat embryo) is alsoprovided, wherein the embryo comprises a donor ES cell that is derivedfrom a non-human animal (e.g., a rodent, e.g., a mouse or a rat) asdescribed herein. In one aspect, the embryo comprises an ES donor cellthat comprises the chimeric MHC I gene, and host embryo cells.

Also provided is a tissue, wherein the tissue is derived from anon-human animal (e.g., a mouse or a rat) as described herein, andexpresses the chimeric MHC I polypeptide (e.g., HLA-A2/H-2Kpolypeptide).

In addition, a non-human cell isolated from a non-human animal asdescribed herein is provided. In one embodiment, the cell is an ES cell.In one embodiment, the cell is an antigen-presenting cell, e.g.,dendritic cell, macrophage, B cell. In one embodiment, the cell is animmune cell. In one embodiment, the immune cell is a lymphocyte.

Also provided is a non-human cell comprising a chromosome or fragmentthereof of a non-human animal as described herein. In one embodiment,the non-human cell comprises a nucleus of a non-human animal asdescribed herein. In one embodiment, the non-human cell comprises thechromosome or fragment thereof as the result of a nuclear transfer.

In one aspect, a non-human induced pluripotent cell comprising geneencoding a chimeric MHC I polypeptide (e.g., HLA-A2/H-2K polypeptide) asdescribed herein is provided. In one embodiment, the induced pluripotentcell is derived from a non-human animal as described herein.

In one aspect, a hybridoma or quadroma is provided, derived from a cellof a non-human animal as described herein. In one embodiment, thenon-human animal is a mouse or rat.

Also provided is a method for making a genetically engineered non-humananimal (e.g., a genetically engineered rodent, e.g., a mouse or a rat)described herein. The method for making a genetically engineerednon-human animal results in the animal whose genome comprises anucleotide sequence encoding a chimeric MHC I polypeptide. In oneembodiment, the method results in a genetically engineered mouse, whosegenome comprises at an endogenous MHC I locus, e.g., H-2K locus, anucleotide sequence encoding a chimeric human/mouse MHC I polypeptide,wherein a human portion of the chimeric MHC I polypeptide comprises anextracellular domain of a human HLA-A2 and a mouse portion comprisestransmembrane and cytoplasmic domains of a mouse H-2K. In someembodiments, the method utilizes a targeting construct made usingVELOCIGENE® technology, introducing the construct into ES cells, andintroducing targeted ES cell clones into a mouse embryo usingVELOCIMOUSE® technology, as described in the Examples. In oneembodiment, the ES cells are a mix of 129 and C57BL/6 mouse strains; inanother embodiment, the ES cells are a mix of BALB/c and 129 mousestrains.

Thus, a nucleotide construct used for generating genetically engineerednon-human animals described herein is also provided. In one aspect, thenucleotide construct comprises: 5′ and 3′ non-human homology arms, ahuman DNA fragment comprising human HLA-A gene sequences, and aselection cassette flanked by recombination sites. In one embodiment,the human DNA fragment is a genomic fragment that comprises both intronsand exons of a human HLA-A gene. In one embodiment, the non-humanhomology arms are homologous to a non-human MHC class I locus (e.g., amouse H-2K locus).

In one embodiment, the genomic fragment comprises a human HLA-A leader,an al domain, an a2 domain and an α3 domain coding sequence. In oneembodiment, the human DNA fragment comprises, from 5′ to 3′: an HLA-Aleader sequence, an HLA-A leader/α1 intron, an HLA-A α1 exon, an HLA-Aα1-α2 intron, an HLA-A α2 exon, an HLA-A α2-α3 intron, and an HLA-A α3exon.

A selection cassette is a nucleotide sequence inserted into a targetingconstruct to facilitate selection of cells (e.g., ES cells) that haveintegrated the construct of interest. A number of suitable selectioncassettes are known in the art. Commonly, a selection cassette enablespositive selection in the presence of a particular antibiotic (e.g.,Neo, Hyg, Pur, CM, Spec, etc.). In addition, a selection cassette may beflanked by recombination sites, which allow deletion of the selectioncassette upon treatment with recombinase enzymes. Commonly usedrecombination sites are IoxP and Frt, recognized by Cre and Flp enzymes,respectively, but others are known in the art.

In one embodiment, the selection cassette is located at the 5′ end thehuman DNA fragment. In another embodiment, the selection cassette islocated at the 3′ end of the human DNA fragment. In another embodiment,the selection cassette is located within the human DNA fragment. Inanother embodiment, the selection cassette is located within an intronof the human DNA fragment. In another embodiment, the selection cassetteis located within the α2-α3 intron.

In one embodiment, the 5′ and 3′ non-human homology arms comprisegenomic sequence at 5′ and 3′ locations of an endogenous non-human(e.g., murine) MHC class I gene locus, respectively (e.g., 5′ of thefirst leader sequence and 3′ of the α3 exon of the non-human MHC Igene). In one embodiment, the endogenous MHC class I locus is selectedfrom mouse H-2K, H-2D and H-2L. In a specific embodiment, the endogenousMHC class I locus is mouse H-2K.

In one aspect, a nucleotide construct is provided, comprising, from 5′to 3′: a 5′ homology arm containing mouse genomic sequence 5′ of theendogenous mouse H-2K locus, a first human DNA fragment comprising afirst genomic sequence of an HLA-A gene, a 5′ recombination sequencesite (e.g., IoxP), a selection cassette, a 3′ recombination sequencesite e.g., IoxP), a second human DNA fragment comprising a secondgenomic sequence of an HLA-A gene and a 3′ homology arm containing mousegenomic sequence 3′ of an endogenous H-2K α3 exon. In one embodiment,the nucleotide construct comprises, from 5′ to 3′: a 5′ homology armcontaining mouse genomic sequence 5′ of the endogenous mouse H-2K locus,a human genomic sequence including an HLA-A leader, an HLA-A leader/α1intron sequence, an HLA-A α1 exon, an HLA-A α1-α2 intron, an HLA-A α2exon, a first 5′ portion of an α2-α3 intron, a selection cassetteflanked by recombination sites, a second 3′ portion of an α2-α3 intron,an HLA-A α3 exon, and a 3′ homology arm containing non-mouse genomicsequence 3′ of the endogenous mouse H-2K α3 exon. In one embodiment, a5′ homology arm sequence is set forth in SEQ ID NO:1, and a 3′ homologyarm sequence is set forth in SEQ ID NO:2.

Upon completion of gene targeting, ES cells or genetically modifiednon-human animals are screened to confirm successful incorporation ofexogenous nucleotide sequence of interest or expression of exogenouspolypeptide. Numerous techniques are known to those skilled in the art,and include (but are not limited to) Southern blotting, long PCR,quantitative PCT (e.g., real-time PCR using TAQMAN®), fluorescence insitu hybridization, Northern blotting, flow cytometry, Western analysis,immunocytochemistry, immunohistochemistry, etc. In one example,non-human animals (e.g., mice) bearing the genetic modification ofinterest can be identified by screening for loss of mouse allele and/orgain of human allele using a modification of allele assay described inValenzuela et al. (2003) High-throughput engineering of the mouse genomecoupled with high-resolution expression analysis, Nature Biotech.21(6):652-659. Other assays that identify a specific nucleotide or aminoacid sequence in the genetically modified animals are known to thoseskilled in the art.

The disclosure also provides a method of modifying an MHC I locus of anon-human animal to express a chimeric human/non-human MHC I polypeptidedescribed herein. In one embodiment, the invention provides a method ofmodifying an MHC I locus of a mouse to express a chimeric human/mouseMHC I polypeptide wherein the method comprises replacing at anendogenous MHC I locus a nucleotide sequence encoding a peptide bindingdomain of a mouse MHC polypeptide with a nucleotide sequence encoding apeptide binding domain of a human MHC I polypeptide. In some aspects, anucleotide sequence of an extracellular domain of a mouse MHC I isreplaced by a nucleotide sequence of an extracellular domain of a humanMHC I. The mouse may fail to express the peptide binding or theextracellular domain of the mouse MHC I from an endogenous MHC I locus.In some embodiments, a nucleotide sequence of an extracellular domain ofa mouse H-2K is replaced by a nucleotide sequence of an extracellulardomain of a human HLA-A2, such that the modified mouse MHC I locusexpresses a chimeric HLA-A2/H-2K polypeptide.

In one aspect, a method for making a chimeric human HLA classI/non-human MHC class I molecule is provided, comprising expressing in asingle cell a chimeric HLA-NH-2K protein from a nucleotide construct,wherein the nucleotide construct comprises a cDNA sequence that encodesan α1, α2, and α3 domain of an HLA-A protein and a transmembrane andcytoplasmic domain of a non-human H-2K protein, e.g., mouse H-2Kprotein. In one embodiment, the nucleotide construct is a viral vector;in a specific embodiment, the viral vector is a lentiviral vector. Inone embodiment, the cell is selected from a CHO, COS, 293, HeLa, and aretinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™cell).

In one aspect, a cell that expresses a chimeric human HLA ClassI/non-human MHC I protein (e.g., HLA-A/H-2K protein) is provided. In oneembodiment, the cell comprises an expression vector comprising achimeric MHC class I gene, wherein the chimeric MHC class I genecomprises a sequence of a human HLA-A gene fused in operable linkagewith a sequence of a non-human H-2K gene, e.g., mouse H-2K gene. In oneembodiment, the sequence of the human HLA-A gene comprises the exonsthat encode α1, α2 and α3 domains of an HLA-A protein. In oneembodiment, the sequence of the non-human H-2K gene comprises the exonsthat encode transmembrane and cytoplasmic domains of an H-2K protein. Inone embodiment, the cell is selected from CHO, COS, 293, HeLa, and aretinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™cell).

A chimeric MHC class I molecule made by a non-human animal as describedherein is also provided, wherein the chimeric MHC class I moleculecomprises α1, α2, and α3 domains from a human HLA-A protein andtransmembrane and cytoplasmic domains from a non-human, e.g., mouse,H-2K protein. The chimeric MHC I polypeptide described herein maybedetected by anti-HLA-A antibodies. Thus, a cell displaying chimerichuman/non-human MHC I polypeptide may be detected and/or selected usinganti-HLA-A antibody. In some instances, the chimeric MHC I polypeptidedescribed herein maybe detected by an anti-HLA-A2 antibody.

Although the following Examples describe a genetically engineered animalwhose genome comprises a replacement of a nucleotide sequence encodingan extracellular domain of mouse H-2K polypeptide with the sequenceencoding an extracellular domain of a human HLA-A at the endogenousmouse H-2K locus, one skilled in the art would understand that a similarstrategy may be used to replace other mouse MHC I loci (H-2D, H-2L,etc.) with their corresponding human HLA loci (HLA-B, HLA-C, etc.).Thus, a non-human animal comprising in its genome a nucleotide sequenceencoding a chimeric human/non-human MHC I polypeptide wherein a humanportion of the polypeptide is derived from another HLA class I proteinis also provided. The replacement of multiple MHC I loci is alsocontemplated.

Genetically Modified β2 Microglobulin Animals

The invention generally provides genetically modified non-human animalsthat comprise in their genome a nucleotide sequence encoding a human orhumanized β2 microglobulin polypeptide; thus, the animals express ahuman or humanized β2 microglobulin polypeptide.

β2 microglobulin or the light chain of the MHC class I complex (alsoabbreviated “β2M”) is a small (12 kDa) non-glycosylated protein, thatfunctions primarily to stabilize the MHC I α chain. The human β2microglobulin gene encodes a protein of 119 amino acids, with 20N-terminal amino acids encoding a leader sequence. The mature proteincomprises 99 amino acids. The gene contains 4 exons, with the first exoncontaining the 5′ untranslated region, the entire leader sequence andthe first two amino acids of the mature polypeptide; the second exonencoding the majority of the mature protein; the third exon encoding thelast four amino acids of the mature protein and a stop codon; and thefourth exon containing the 3′ non-translated region. Gussow et al.(1987) The β2-Microglobulin Gene. Primary Structure and Definition ofthe Transcriptional Unit, J. Immunol. 139:3131-38. β2 microglobulin isnon-covalently associated with MHC I. Unbound β2 microglobulin is foundin body fluids, such as plasma, and is carried to the kidney forexcretion. Kidney dysfunction causes accumulation of β2 microglobulin,which can be pathogenic (e.g., Dialysis Related Amyloidosis); theaccumulated protein forms filamentous fibrils resembling amyloid plaquesin joints and connective tissues.

In addition to Dialysis Related Amyloidosis, β2 microglobulin has beenimplicated in a number of other disorders. Elevated levels of β2microglobulin were detected in lymphocytic malignancies, e.g.,non-Hodgkin's lymphoma and multiple myeloma. See, e.g., Shi et al.(2009) β2 Microglobulin: Emerging as a Promising Cancer TherapeuticTarget, Drug Discovery Today 14:25-30. Some other malignancies withelevated levels of β2 microglobulin include breast cancer, prostatecancer, lung cancer, renal cancer, gastrointestinal and nasopharyngealcancers. Overexpression of β2 microglobulin has been suggested to havetumor growth promoting effects. Id. It has also been recently shown thatβ2 microglobulin drives epithelial to mesenchymal transition, promotingcancer bone and soft tissue metastasis in breast, prostate, lung andrenal cancers. Josson et al. (2011) β2 microglobulin Induces Epitelialto Mesenchymal Transition and Confers Cancer Lethality and BoneMetastasis in Human Cancer Cells, Cancer Res. 71(7): 1-11. β2microglobulin interacts with a non-classical MHC I member,hemochromatosis (HFE) protein, and with the transferrin receptor, andmodulates iron homeostasis. Id. Involvement of β2 microglobulin in otherhallmarks of malignancy (self-renewal, angiogenesis enhancement,resistance to treatment) is widely documented in the art.

Mice deficient in β2 microglobulin have been reported. See, Koller etal. (1990) Normal development of mice deficient in β2 m, MHC class Iproteins, and CD8+ T cells, Science 248: 1227-1230, As reported inKoller et al., these mice appeared healthy, however, MHC class Iexpression was not detected. Further, most T cell populations appearednormal in some tissues, while a marked decrease of CD8+ T cells wasobserved in others. This purported lack of MHC I expression disagreeswith previous results obtained by Allen et al. ((1986) β2 microglobulinIs Not Required for Cell Surface Expression of the Murine Class IHistocompatibility Antigen H-2D^(b) or of a Truncated H-2D^(b), Proc.Natl. Acad, Sci, USA 83:7447-7451). Allen et al. reported that β2microglobulin was not absolutely required for cell surface expression ofall MHC I complexes, because cells lacking β2 microglobulin were able toexpress H-2D^(b). However, the function of H-2D^(b) in these cells waspresumably compromised, and conformation of H-2D^(b) was different fromthe native protein, which explains the inability of Koller andcolleagues to detect this protein using antibodies against nativeH-2D^(b). However, cells lacking β₂ microglobulin can reportedly presentendogenous antigen to CD8+ T cells (including exogenous CD8+ T cellsfrom normal mice), and β2 microglobulin is reportedly not required inorder to develop high levels of H-2^(d) MHC class I-restricted CD8+ CTLsin response to antigen challenge in mice, although it is required inorder to sustain an effective immune response. Quinn et al. (1997)Virus-Specific, CD8+ Major Histocompatibility Complex Class I-RestrictedCytotoxic T Lymphocytes in Lymphocytic Choriomeningitis Virus-Infectedβ2-Microglobulin-Deficient Mice, J. Virol. 71:8392-8396. It is of notethat the ability to generate high levels of such T cells in the absenceof β2 microglobulin is reportedly limited to an H-2^(d) MHC classI-restricted response. β2 microglobulin deficient mice have beenreported to have a host of dramatic characteristics, such as, forexample, an increased susceptibility to some parasitic diseases, anincreased susceptibility to hepatitis infections, a deficiency in ironmetabolism, and an impaired breeding phenotype. Cooper et al. (2007) Animpaired breeding phenotype in mice with a genetic deletion of Beta-2microglobulin and diminished MHC class I expression: Role inreproductive fitness, Biol. Reprod. 77:274-279.

Mice that express human β2 microglobulin as well as human HLA class Imolecules (i.e., HLA-B7) on a randomly inserted transgene have beenreported. Chamberlain et al. (1988) Tissue-specific and cell surfaceexpression of human major histocompatibility complex class I heavy(HLA-B7) and light (β2-microglobulin) chain genes in transgenic mice,Proc. Natl. Acad. Sci. USA 85:7690-7694. The expression of human HLAclass I was consistent with that of endogenous class I with a markeddecrease in the liver. Id. The expression of human β2 microglobulin wasalso consistent with the endogenous β2 microglobulin, while expressionof the human HLA class I molecule was increased 10- to 17-fold in doubletransgenic mice. Id. However, the authors did not attempt a replacementof a mouse endogenous β2 microglobulin locus with a human β2microglobulin locus.

Therefore, disclosed herein is a genetically engineered non-human animal(e.g., a rodent, e.g., a mouse or a rat) whose genome comprises anucleotide sequence encoding a human or humanized β2 microglobulinpolypeptide. In one aspect, the animal does not express an endogenousnon-human β2 microglobulin from an endogenous non-human β2 microglobulinlocus. In some embodiments, the nucleotide sequence encodes a β2microglobulin polypeptide that is partially human and partiallynon-human, e.g., it contains some amino acids that correspond to humanand some amino acids that correspond to non-human β2 microglobulin. Inone aspect, the non-human animal does not express an endogenousnon-human β2 microglobulin polypeptide from an endogenous non-humanlocus, and only expresses the human or humanized β2 microglobulinpolypeptide. In one example, the non-human animal does not express acomplete endogenous non-human β2 microglobulin polypeptide but onlyexpresses a portion of a non-human endogenous β2 microglobulinpolypeptide from an endogenous β2 microglobulin locus. Thus, in variousembodiments, the animal does not express a functional non-human β2microglobulin polypeptide from an endogenous non-human β2 microglobulinlocus. In a specific aspect, the nucleotide sequence encoding the humanor humanized β2 microglobulin is located at an endogenous non-human β2microglobulin locus. In one aspect, the animal comprises two copies ofβ2 microglobulin locus comprising a nucleotide sequence encoding a humanor humanized β2 microglobulin polypeptide. In another aspect, the animalcomprises one copy of β2 microglobulin locus comprising a nucleotidesequence encoding a human or humanized β2 microglobulin polypeptide.Thus, the animal may be homozygous or heterozygous for β2 microglobulinlocus comprising a nucleotide sequence that encodes a human or humanizedβ2 microglobulin polypeptide. The nucleotide sequence of the human orhumanized β2 microglobulin may be derived from a collection of β2microglobulin sequences that are naturally found in human populations.In various embodiments, the genetically engineered non-human animal ofthe invention comprises in its germline a nucleotide sequence encoding ahuman or humanized β2 microglobulin. In one embodiment, a nucleotidesequence encoding a human or humanized β2 microglobulin polypeptidecomprises a nucleotide sequence encoding a polypeptide comprising ahuman β2 microglobulin amino acid sequence. In one embodiment, thepolypeptide is capable of binding to an MHC I protein.

The nucleotide sequence encoding the human or humanized β2 microglobulinpolypeptide may comprise nucleic acid residues corresponding to theentire human β2 microglobulin gene. Alternatively, the nucleotidesequence may comprise nucleic acid residues encoding amino acid sequenceset forth in amino acids 21-119 of a human β2 microglobulin protein(i.e., amino acid residues corresponding to the mature human β2microglobulin). In an alternative embodiment, the nucleotide sequencemay comprise nucleic acid residues encoding amino acid sequence setforth in amino acids 23-115 of a human β2 microglobulin protein, forexample, amino acid sequence set forth in amino acids 23-119 of a humanβ2 microglobulin protein. The nucleic and amino acid sequences of humanβ2 microglobulin are described in Gussow et al., supra, incorporatedherein by reference.

Thus, the human or humanized β2 microglobulin polypeptide may compriseamino acid sequence set forth in amino acids 23-115 of a human β2microglobulin polypeptide, e.g., amino acid sequence set forth in aminoacids 23-119 of a human β2 microglobulin polypeptide, e.g., amino acidsequence set forth in amino acids 21-119 of a human β2 microglobulinpolypeptide. Alternatively, the human β2 microglobulin may compriseamino acids 1-119 of a human β2 microglobulin polypeptide.

In some embodiments, the nucleotide sequence encoding a human orhumanized β2 microglobulin comprises a nucleotide sequence set forth inaxon 2 to exon 4 of a human β2 microglobulin gene. Alternatively, thenucleotide sequence comprises nucleotide sequences set forth in axons 2,3, and 4 of a human β2 microglobulin gene. In this embodiment, thenucleotide sequences set forth in exons 2, 3, and 4 are operably linkedto allow for normal transcription and translation of the gene. Thus, inone embodiment, the human sequence comprises a nucleotide sequencecorresponding to exon 2 to exon 4 of a human β2 microglobulin gene. In aspecific embodiment, the human sequence comprises a nucleotide sequencecorresponding to exon 2 to about 267 bp after exon 4 of a human β2microglobulin gene. In a specific embodiment, the human sequencecomprises about 2.8 kb of a human β2 microglobulin gene.

Thus, the human or humanized β2 microglobulin polypeptide may be encodedby a nucleotide sequence comprising nucleotide sequence set forth inexon 2 to axon 4 of a human β2 microglobulin, e.g., nucleotide sequencecorresponding to exon 2 to exon 4 of a human β2 microglobulin gene.Alternatively, the polypeptide may be encoded by a nucleotide sequencecomprising nucleotide sequences set forth in exons 2, 3, and 4 of ahuman β2 microglobulin gene. In a specific embodiment, the human orhumanized β2 microglobulin polypeptide is encoded by a nucleotidesequence corresponding to axon 2 to about 267 bp after axon 4 of a humanβ2 microglobulin gene. In another specific embodiment, the human orhumanized polypeptide is encoded by a nucleotide sequence comprisingabout 2.8 kb of a human β2 microglobulin gene. As exon 4 of the β2microglobulin gene contains the 5′ untranslated region, the human orhumanized polypeptide may be encoded by a nucleotide sequence comprisingexons 2 and 3 of the β2 microglobulin gene.

It would be understood by those of ordinary skill in the art thatalthough specific nucleic acid and amino acid sequences to generategenetically engineered animals are described in the present examples,sequences of one or more conservative or non-conservative amino acidsubstitutions, or sequences differing from those described herein due tothe degeneracy of the genetic code, are also provided.

Therefore, a non-human animal that expresses a human β2 microglobulinsequence is provided, wherein the β2 microglobulin sequence is at leastabout 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human β2microglobulin sequence. In a specific embodiment, the β2 microglobulinsequence is at least about 90%, 95%, 96%. 97%, 98%, or 99% identical tothe human β2 microglobulin sequence described in the Examples. In oneembodiment, the human β2 microglobulin sequence comprises one or moreconservative substitutions. In one embodiment, the human β2microglobulin sequence comprises one or more non-conservativesubstitutions.

In addition, provided are non-human animals wherein the nucleotidesequence encoding a human or humanized β2 microglobulin protein alsocomprises a nucleotide sequence set forth in exon 1 of a non-human β2microglobulin gene. Thus, in a specific embodiment, the non-human animalcomprises in its genome a nucleotide sequence encoding a human orhumanized β2 microglobulin wherein the nucleotide sequence comprisesexon 1 of a non-human β2 microglobulin and exons 2, 3, and 4 of a humanβ2 microglobulin gene. Thus, the human or humanized β2 microglobulinpolypeptide is encoded by exon 1 of a non-human β2 microglobulin geneand exons 2, 3, and 4 of a human β2 microglobulin gene (e.g., exons 2and 3 of a human β2 microglobulin gene).

Similarly to a non-human animal comprising a nucleotide sequenceencoding a chimeric human/non-human MHC I polypeptide, the non-humananimal comprising a nucleotide sequence encoding a human or humanized β2microglobulin may be selected from a group consisting of a mouse, rat,rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat,chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). Insome embodiments of the invention, the non-human animal is a mammal. Ina specific embodiment, the non-human animal is a murine, e.g., a rodent(e.g., a mouse or a rat). In one embodiment, the animal is a mouse.

Thus, in some aspects, a genetically engineered mouse is provided,wherein the mouse comprises a nucleotide sequence encoding a human or ahumanized β2 microglobulin polypeptide as described herein. Agenetically engineered mouse is provided, wherein the mouse comprises atits endogenous β2 microglobulin locus a nucleotide sequence encoding ahuman or humanized β2 microglobulin polypeptide (e.g., a human orsubstantially human β2 microglobulin polypeptide). In some embodiments,the mouse does not express an endogenous β2 microglobulin polypeptide(e.g., a functional endogenous β2 microglobulin polypeptide) from anendogenous β2 microglobulin locus. In some embodiments, the geneticallyengineered mouse comprises a nucleotide sequence comprising exon 1 of amouse β2 microglobulin gene and exons 2, 3, and 4 of a human β2microglobulin gene. In some embodiments, the mouse expresses the humanor humanized β2 microglobulin polypeptide.

In one aspect, a modified non-human β2 microglobulin locus is providedthat comprises a heterologous β2 microglobulin sequence. In oneembodiment, the heterologous β2 microglobulin sequence is a human or ahumanized sequence.

In one embodiment, the modified locus is a rodent locus. In a specificembodiment, the rodent locus is selected from a mouse or rat locus. Inone embodiment, the non-human locus is modified with at least one humanβ2 microglobulin coding sequence.

In one embodiment, the heterologous β2 microglobulin sequence isoperably linked to endogenous regulatory elements, e.g., endogenouspromoter and/or expression control sequence. In a specific embodiment,the heterologous β2 microglobulin sequence is a human sequence and thehuman sequence is operably linked to an endogenous promoter and/orexpression control sequence.

In one aspect, a modified non-human β2 microglobulin locus is providedthat comprises a human sequence operably linked to an endogenouspromoter and/or expression control sequence.

In various aspects, the human or humanized β2 microglobulin expressed bya genetically modified non-human animal, or cells, embryos, or tissuesderived from a non-human animal, preserves all the functional aspects ofthe endogenous and/or human β2 microglobulin. For example, it ispreferred that the human or humanized β2 microglobulin binds the a chainof MHC I polypeptide (e,g., endogenous non-human or human MHC Ipolypeptide). The human or humanized β2 microglobulin polypeptide maybind, recruit or otherwise associate with any other molecules, e.g.,receptor, anchor or signaling molecules that associate with endogenousnon-human and/or human β2 microglobulin (e.g., HFE, etc.).

In addition to genetically modified animals (e.g., rodents, e.g., miceor rats), also provided is a tissue or cell, wherein the tissue or cellis derived from a non-human animal as described herein, and comprises aheterologous β2 microglobulin gene or β2 microglobulin sequence, i.e.,nucleotide and/or amino acid sequence. In one embodiment, theheterologous β2 microglobulin gene or β2 microglobulin sequence is ahuman or humanized β2 microglobulin gene or human or humanized β2microglobulin sequence. Preferably, the cell is a nucleated cell. Thecell may be any cell known to express MHC I complex, e.g., an antigenpresenting cell. The human or humanized β2 microglobulin polypeptideexpressed by said cell may interact with endogenous non-human MHC I(e.g., rodent MHC I), to form a functional MHC I complex. The resultantMHC I complex may be capable of interacting with a T cell, e.g., acytotoxic T cell Thus, also provided is an in vitro complex of a cellfrom a non-human animal as described herein and a T cell.

Also provided are non-human cells that comprise human or humanized β2microglobulin gene or sequence, and an additional human or humanizedsequence, e.g., chimeric MHC I polypeptide presently disclosed. In suchan instance, the human or humanized β2 microglobulin polypeptide mayinteract with, e.g., a chimeric human/non-human MHC I polypeptide, and afunctional MHC I complex may be formed. In some aspects, such complex iscapable of interacting with a TCR on a T cell, e.g., a human or anon-human T cell. Thus, also provided in an in vitro complex of a cellfrom a non-human animal as described herein and a human or a non-human Tcell.

Another aspect of the disclosure is a rodent embryo (e.g., a mouse or arat embryo) comprising a heterologous β2 microglobulin gene or β2microglobulin sequence as described herein. In one embodiment, theembryo comprises an ES donor cell that comprises the heterologous β2microglobulin gene or β2 microglobulin sequence, and host embryo cells.The heterologous β2 microglobulin gene or β2 microglobulin sequence is ahuman or humanized β2 microglobulin gene or β2 microglobulin sequence.

This invention also encompasses a non-human cell comprising a chromosomeor fragment thereof of a non-human animal as described herein (e.g.,wherein the chromosome or fragment thereof comprises a nucleotidesequence encoding a human or humanized β2 microglobulin polypeptide).The non-human cell may comprise a nucleus of a non-human animal asdescribed herein. In one embodiment, the non-human cell comprises thechromosome or fragment thereof as the result of a nuclear transfer.

In one aspect, a non-human induced pluripotent cell comprising aheterologous β2 microglobulin gene or β2 microglobulin sequence isprovided. In one embodiment, the induced pluripotent cell is derivedfrom a non-human animal as described herein. In one embodiment, theheterologous β2 microglobulin gene or β2 microglobulin sequence is ahuman or humanized gene or sequence.

Also provided is a hybridoma or quadroma, derived from a cell of anon-human animal as described herein. In one embodiment, the non-humananimal is a mouse or rat.

The disclosure also provides methods for making a genetically engineerednon-human animal (e.g., a genetically engineered rodent, e.g., a mouseor a rat) described herein. The methods result in an animal whose genomecomprises a nucleotide sequence encoding a human or humanized β2microglobulin polypeptide. In one aspect, the methods result in agenetically engineered mouse, whose genome comprises at an endogenous β2microglobulin locus a nucleotide sequence encoding a human or humanizedβ2 microglobulin polypeptide. In some instances, the mouse does notexpress a functional mouse β2 microglobulin from an endogenous mouse β2microglobulin locus. In some aspects, the methods utilize a targetingconstruct made using VELOCIGENE® technology, introducing the constructinto ES cells, and introducing targeted ES cell clones into a mouseembryo using VELOCIMOUSE® technology, as described in the Examples. Inone embodiment, the ES cells are mix of 129 and C57BL/6 mouse strains;in another embodiment, the ES cells are a mix of BALB/c and 129 mousestrains.

Also provided is a nucleotide construct used for generating geneticallyengineered non-human animals. The nucleotide construct may comprise: 5′and 3′ non-human homology arms, a human DNA fragment comprising human β2microglobulin sequences, and a selection cassette flanked byrecombination sites. In one embodiment, the human DNA fragment is agenomic fragment that comprises both introns and exons of a human β2microglobulin gene. In one embodiment, the non-human homology arms arehomologous to a non-human β2 microglobulin locus. The genomic fragmentmay comprise exons 2, 3, and 4 of the human β2 microglobulin gene. Inone instance, the genomic fragment comprises, from 5′ to 3′: exon 2,intron, exon 3, intron, and exon 4, all of human β2 microglobulinsequence. The selection cassette may be located anywhere in theconstruct outside the β2 microglobulin coding region, e.g., it may belocated 3′ of exon 4 of the human β2 microglobulin. The 5′ and 3′non-human homology arms may comprise genomic sequence 5′ and 3′ ofendogenous non-human β2 microglobulin gene, respectively. In anotherembodiment, the 5′ and 3′ non-human homology arms comprise genomicsequence 5′ of exon 2 and 3′ of exon 4 of endogenous non-human gene,respectively.

Another aspect of the invention relates to a method of modifying a β2microglobulin locus of a non-human animal (e.g., a rodent, e.g., a mouseor a rat) to express a human or humanized β2 microglobulin polypeptidedescribed herein. One method of modifying a β2 microglobulin locus of amouse to express a human or humanized β2 microglobulin polypeptidecomprises replacing at an endogenous β2 microglobulin locus a nucleotidesequence encoding a mouse β2 microglobulin with a nucleotide sequenceencoding the human or humanized β2 microglobulin polypeptide. In oneembodiment of such method, the mouse does not express a functional β2microglobulin polypeptide from an endogenous mouse β2 microglobulinlocus. In some specific embodiments, the nucleotide sequence encodingthe human or humanized β2 microglobulin polypeptide comprises nucleotidesequence set forth in exons 2 to 4 of the human β2 microglobulin gene.In other embodiments, the nucleotide sequence encoding the human orhumanized β2 microglobulin polypeptide comprises nucleotide sequencesset forth in exons 2, 3, and 4 of the human β2 microglobulin gene.

Genetically Modified MHC I/β2 Microglobulin Animals

In various embodiments, the invention generally provides geneticallymodified non-human animals that comprise in their genome nucleotidesequences encoding both human or humanized MHC I and β2 microglobulinpolypeptides; thus, the animals express both human or humanized MHC Iand β2 microglobulin polypeptides.

Functional differences arise in the use of mixed human/non-human systemcomponents. HLA class I binds β2 microglobulin tighter than mouse classI. Bernabeu (1984) β2-microgobulin from serum associates with MHC classI antigens on the surface of cultured cells, Nature 308:642-645.Attempts to abrogate functional differences are reflected in theconstruction of particular humanized MHC mice. H-2 class I and class 2knockout mice (in a mouse β2 microglobulin KO background) that express arandomly integrated human HLA-A2.1/HLA-DR1 chimeric transgene having anα1 and α2 of human HLA-A2.1, and α3 of mouse H-2D^(b), attached at itsN-terminal via a linker to the C-terminus of human β2-microglobulin havebeen developed. See, e.g., Pajot et al. (2004) A mouse model of humanadaptive immune functions: HLA-A2.1-/HLA-DR1-transgenic H-2 classI-/class II-knockout mice, Eur. J. Immunol. 34:3060-3069. These micereportedly generate antigen-specific antibody and CTL responses againsthepatitis B virus, whereas mice merely transgenic for HLA-A2.1 or H-2class I/class II knockout mice do not. The deficiency of mice that aremerely transgenic for the genes presumably stems from the ability ofsuch mice to employ endogenous class I and/or class II genes tocircumvent any transgene, an option not available to MHC knockout mice.However, the mice may express at least H-2D^(b), presumably due tobreedings into the mouse β2 microglobulin knockout mouse background(see, Pajot et al., supra; which apparently comprised an intactendogenous class I and class II locus).

Cell surface expression of the chimeric fusion with human β2microglobulin is reportedly lower than endogenous MHC expression, butsurvivability/rate of NK killing is not reported, nor is the rate of NKself-killing. Pajot et al., supra. Some improvement in CD8+ T cellnumbers was observed over MHC class I-deficient β2-microglobulinknockout mice (2-3% of total splenocytes, vs. 0.6-1% in the β2 KO mice).However, T cell variable region usage exhibited altered profiles for BV5.1, BV 5,2, and BV 11 gene segments. Both CD8+ and CD4+ T cellresponses were reportedly restricted to the appropriate hepatitis Bantigen used to immunize the mice, although at least two mice killedcells bearing either of the antigens, where the mice were immunized withonly one antigen, which might be due to a lack of NK cell inhibition orlack of NK cell selectivity.

As mentioned above, mice transgenic for both human MHC I and human β2microglobulin comprise a nucleotide sequence encoding a chimeric MHCI/β2 microglobulin protein, wherein the MHC I and β2 microglobulinportions are contained within a single polypeptide chain, resulting inMHC I α chain and β2 microglobulin being covalently linked to each otherand thereby tethered at the cell surface. A mouse which comprises in itsgenome two independent nucleotide sequences, one encoding a human orhumanized MHC I polypeptide and the other encoding a human or humanizedβ2 microglobulin polypeptide is provided. The mouse provided hereinwould express an MHC I complex that more closely resembles an MHC Icomplex present in nature, wherein MHC I a chain and β2 microglobulinare provided on two separate polypeptide chains with β2 microglobulinnon-covalently associating with the MHC I α chain.

Thus, the present disclosure provides a non-human animal comprising inits genome: a first nucleotide sequence encoding a human or humanizedMHC I polypeptide, and a second nucleotide sequence encoding a human orhumanized β2 microglobulin polypeptide. In one aspect, provided is anon-human animal comprising in its genome: (a) a first nucleotidesequence encoding a chimeric human/non-human MHC I polypeptide, whereinthe human portion of the chimeric polypeptide comprises a peptidebinding domain or an extracellular domain of a human MHC I (e,g., HLA-A,HLA-B, or HLA-C; e.g., HLA-A2), and (b) a second nucleotide sequenceencoding a human or humanized β2 microglobulin polypeptide.

The first nucleotide sequence may be located at an endogenous non-humanMHC I locus such that the animal comprises in its genome a replacementat the MHC I locus of all or a portion of endogenous MHC I gene (e.g., aportion encoding a peptide binding domain or an extracellular domain)with the corresponding human MHC I sequence. Thus, the animal maycomprise at an endogenous MHC I locus a nucleotide sequence encoding anextracellular domain of a human MHC I (e.g., HLA-A, HLA-B, or HLA-C;e.g., HLA-A2) and transmembrane and cytoplasmic domains of endogenousnon-human MHC I (e.g., H-2K, H-2D, etc., e.g., H-2Kb). In one aspect,the animal is a mouse, and the first nucleotide sequence comprises anucleotide sequence encoding an extracellular domain of a human HLA-A2(e.g., HLA-A2.1) and transmembrane and cytoplasmic domains of a mouseH-2K (e.g., H-2Kb).

The second nucleotide sequence may be located at an endogenous non-humanβ2 microglobulin locus such that the animal comprises in its genome areplacement at the β2 microglobulin locus of all or a portion ofendogenous β2 microglobulin gene with the corresponding human β2microglobulin sequence. The second nucleotide sequence may comprise anucleotide sequence set forth in exon 2 to exon 4 of a human β2microglobulin gene. Alternatively, the second nucleotide sequence maycomprise nucleotide sequences set forth in exons 2, 3, and 4 of a humanβ2 microglobulin gene. In this embodiment, nucleotide sequences areoperably linked to each other. The second nucleotide sequence mayfurther comprise the sequence of exon 1 of a non-human β2 microglobulingene.

In one aspect, the animal does not express a functional MHC I from anendogenous non-human MHC I locus (e.g., does not express either apeptide binding domain or an extracellular domain of the endogenous MHCI), and the animal does not express a functional β2 microglobulinpolypeptide from an endogenous non-human β2 microglobulin locus. In someaspects, the animal is homozygous for both an MHC I locus comprising anucleotide sequence encoding a chimeric human/non-human MHC Ipolypeptide and a β2 microglobulin locus comprising a nucleotidesequence encoding a human or humanized β2 microglobulin. In otheraspects, the animal is heterozygous for both an MHC I locus comprising anucleotide sequence encoding a chimeric human/non-human MHC Ipolypeptide and a β2 microglobulin locus comprising a nucleotidesequence encoding a human or humanized β2 microglobulin.

Preferably, the first and the second nucleotide sequences are operablylinked to endogenous expression control elements (e.g., promoters,enhancers, silencers, etc.).

Various other embodiments of the first and second nucleotide sequences(and the polypeptides they encode) encompassed herein may be readilyunderstood from the embodiments described throughout the specification,e,g., those described in the sections related to genetically engineeredMHC I animals and genetically engineered β2 microglobulin animals.

In one aspect, the disclosure provides a mouse comprising in its genome(a) a first nucleotide sequence encoding a chimeric human/mouse MHC Ipolypeptide (specifically, HLA-A2/H-2K polypeptide), wherein the humanportion of the chimeric polypeptide comprises an extracellular domain ofa human HLA-A2 and the mouse portion comprises transmembrane andcytoplasmic domains of a mouse H-2K, and (b) a second nucleotidesequence encoding a human or humanized β2 microglobulin polypeptide(e.g., wherein the nucleotide sequence comprises a nucleotide sequenceset forth in exon 2 to exon 4 of the human β2 microglobulin gene ornucleotide sequences set forth in exon 2, 3, and 4 of the human β2microglobulin gene), wherein the first nucleotide sequence is located atan endogenous H-2K locus, and the second sequence is located at anendogenous β2 microglobulin locus. In one embodiment, the mouse does notexpress functional H-2K and mouse β2 microglobulin polypeptides fromtheir respective endogenous loci. In one embodiment, the mouse expressesboth the chimeric human/mouse MHC I polypeptide and the human orhumanized β2 microglobulin polypeptide.

As shown in the following Examples, animals genetically engineered toco-express both the human or humanized MHC I and β2 microglobulindisplayed increased expression of chimeric MHC class I on cell surfacein comparison to animals humanized for MHC I alone. In some embodiments,co-expression of human or humanized MHC I and β2 microglobulin increasescell surface expression of human or humanized MHC I by more than about10%, e.g., more than about 20%, e.g., about 50% or more, e.g., about70%, over the expression of human or humanized MHC I in the absence ofhuman or humanized microglobulin.

The disclosure also provides a method of making genetically engineerednon-human animals (e.g., rodents, e.g., rats or mice) whose genomecomprises a first and a second nucleotide sequence as described herein.The method generally comprises generating a first genetically engineerednon-human animal whose genome comprises a first nucleotide sequencedescribed herein (i.e., a human or humanized MHC I sequence), generatinga second genetically engineered non-human animal whose genome comprisesa second nucleotide sequence described herein (i.e., a human orhumanized β2 microglobulin sequence), and breeding the first and thesecond animal to obtain progeny whose genome contains both nucleotidesequences. In one embodiment, the first and the second animal areheterozygous for the first and the second nucleotide sequence,respectively. In one embodiment, the first and the second animal arehomozygous for the first and the second nucleotide sequence,respectively. In one embodiment, the first and second animals aregenerated through replacement of endogenous non-human loci with thefirst and the second nucleotide sequences, respectively. In one aspect,the first and the second animals are generated through utilization ofconstructs generated via VELOCIGENE® technology, and introducingtargeted ES cell clones bearing such constructs into an embryo (e.g., arodent embryo, e,g., a mouse or a rat embryo) via the VELOCIMOUSE®method.

Use of Genetically Modified Animals

In various embodiments, the genetically modified non-human animalsdescribed herein make APCs with human or humanized MHC I and/or β2microglobulin on the cell surface and, as a result, present peptidesderived from cytosolic proteins as epitopes for CTLs in a human-likemanner, because substantially all of the components of the complex arehuman or humanized. The genetically modified non-human animals of theinvention can be used to study the function of a human immune system inthe humanized animal; for identification of antigens and antigenepitopes that elicit immune response (e.g., T cell epitopes, e.g.,unique human cancer epitopes), e.g., for use in vaccine development; foridentification of high affinity T cells to human pathogens or cancerantigens (i.e., T cells that bind to antigen in the context of human MHCI complex with high avidity), e.g., for use in adaptive T cell therapy;for evaluation of vaccine candidates and other vaccine strategies; forstudying human autoimmunity; for studying human infectious diseases; andotherwise for devising better therapeutic strategies based on human MHCexpression.

The MHC I complex binds peptides and presents them on cell surface. Oncepresented on the surface in the context of such a complex, the peptidesare recognizable by T cells. For example, when the peptide is derivedfrom a pathogen or other antigen of interest (e.g., a tumor antigen), Tcell recognition can result in T cell activation, macrophage killing ofcells bearing the presented peptide sequence, and B cell activation ofantibodies that bind the presented sequence.

T cells interact with cells expressing MHC I complex through thepeptide-bound MHC class I ectodomain and the T cell's CD8 ectodomain.CD8+ T cells that encounter APC's that have suitable antigens bound tothe MHC class I molecule will become cytotoxic T cells. Thus, antigensthat in the context of MHC class I bind with high avidity to a T cellreceptor are potentially important in the development of treatments forhuman pathologies. However, presentation of antigens in the context ofmouse MHC I is only somewhat relevant to human disease, since human andmouse MHC complexes recognize antigens differently, e.g., a mouse MHC Imay not recognize the same antigens or may present different epitopesthan a human MHC I. Thus, the most relevant data for human pathologiesis obtained through studying the presentation of antigen epitopes byhuman MHC I.

Thus, in various embodiments, the genetically engineered animals of thepresent invention are useful, among other things, for evaluating thecapacity of an antigen to initiate an immune response in a human, andfor generating a diversity of antigens and identifying a specificantigen that may be used in human vaccine development.

In one aspect, a method for determining antigenicity in a human of apeptide sequence is provided, comprising exposing a genetically modifiednon-human animal as described herein to a molecule comprising thepeptide sequence, allowing the non-human animal to mount an immuneresponse, and detecting in the non-human animal a cell that binds asequence of the peptide presented by a chimeric human/non-human MHC I,or a humanized MHC I complex (comprising a chimeric human/non-human MHCI and a human or humanized β2 microglobulin) as described herein.

In one aspect, a method for determining whether a peptide will provoke acellular immune response in a human is provided, comprising exposing agenetically modified non-human animal as described herein to thepeptide, allowing the non-human animal to mount an immune response, anddetecting in the non-human animal a cell that binds a sequence of thepeptide by a chimeric human/non-human MHC class I molecule as describedherein. In one embodiment, the non-human animal following exposurecomprises an MHC class I-restricted CD8+ cytotoxic T lymphocyte (CTL)that binds the peptide. In one embodiment, the CTL kills a cell bearingthe peptide.

In one aspect, a method for identifying a human CTL epitope is provided,comprising exposing a non-human animal as described herein to an antigencomprising a putative CTL epitope, allowing the non-human animal tomount an immune response, isolating from the non-human animal an MHCclass I-restricted CD8+ CTL that binds the epitope, and identifying theepitope bound by the MHC class I-restricted CD8+ CTL.

In one aspect, a method is provided for identifying an HLA classI-restricted peptide whose presentation by a human cell and binding by ahuman lymphocyte (e.g., human T cell) will result in cytotoxicity of thepeptide-bearing cell, comprising exposing a non-human animal (or MHCclass I-expressing cell thereof) as described herein to a moleculecomprising a peptide of interest, isolating a cell of the non-humananimal that expresses a chimeric human/non-human class I molecule thatbinds the peptide of interest, exposing the cell to a human lymphocytethat is capable of conducting HLA class I-restricted cytotoxicity, andmeasuring peptide-induced cytotoxicity.

In one aspect, a method is provided for identifying an antigen thatgenerates a cytotoxic T cell response in a human, comprising exposing aputative antigen to a mouse as described herein, allowing the mouse togenerate an immune response, and identifying the antigen bound by theHLA-A-restricted molecule.

In one embodiment, the antigen comprises a bacterial or viral surface orenvelope protein. In one embodiment, the antigen comprises an antigen onthe surface of a human tumor cell. In one embodiment, the antigencomprises a putative vaccine for use in a human. In one embodiment, theantigen comprises a human epitope that generates antibodies in a human.In another embodiment, the antigen comprises a human epitope thatgenerates high affinity CTLs that target the epitope/MHC I complex.

In one aspect, a method is provided for determining whether a putativeantigen contains an epitope that upon exposure to a human immune systemwill generate an HLA-A-restricted immune response (e.g.,HLA-A2-restricted response), comprising exposing a mouse as describedherein to the putative antigen and measuring an antigen-specificHLA-A-restricted (e.g., HLA-A2-restricted) immune response in the mouse.

In one embodiment, the putative antigen is selected from abiopharmaceutical or fragment thereof, a non-self protein, a surfaceantigen of a non-self cell, a surface antigen of a tumor cell, a surfaceantigen of a bacterial or yeast or fungal cell, a surface antigen orenvelope protein of a virus.

In addition, the genetically engineered non-human animals describedherein may be useful for identification of T cell receptors, e.g.,high-avidity T cell receptors, that recognize an antigen of interest,e.g., a tumor or another disease antigen. The method may comprise:exposing the non-human animal described herein to an antigen, allowingthe non-human animal to mount an immune response to the antigen,isolating from the non-human animal a T cell comprising a T cellreceptor that binds the antigen presented by a human or humanized MHC I,and determining the sequence of said T cell receptor.

In one aspect, a method for identifying a T cell receptor variabledomain having high affinity for a human tumor antigen is provided;comprising exposing a mouse comprising humanized MHC I α1, α2, and α3domains (e.g., HLA-A2 α1, α2, and α3 domains) to a human tumor antigen;allowing the mouse to generate an immune response; and, isolating fromthe mouse a nucleic acid sequence encoding a T cell receptor variabledomain, wherein the T cell receptor variable domain binds the humantumor antigen with a K_(D) of no higher than about 1 nanomolar.

In one embodiment, the mouse further comprises a replacement at theendogenous mouse T cell receptor variable region gene locus with aplurality of unrearranged human T cell receptor variable region genesegments, wherein the unrearranged human T cell receptor variable regiongene segments recombine to encode a chimeric human-mouse T cell receptorgene comprising a human variable region and a mouse constant region. Inone embodiment, the mouse comprises a human CD8 transgene, and the mouseexpresses a functional human CD8 protein.

T cell receptors having high avidity to tumor antigens are useful incell-based therapeutics. T cell populations with high avidity to humantumor antigens have been prepared by exposing human T cells to HLA-A2that has been mutated to minimize CD8 binding to the α3 subunit, inorder to select only those T cells with extremely high avidity to thetumor antigen (i.e., T cell clones that recognize the antigen in spiteof the inability of CD8 to bind α3). See, Pittet et al. (2003) α3 DomainMutants of Peptide/MHC Class I Multimers Allow the Selective Isolationof High Avidity Tumor-Reactive CD8 T Cells, J. Immunol. 171:1844-1849.The non-human animals, and cells of the non-human animals, are usefulfor identifying peptides that will form a complex with human HLA class Ithat will bind with high avidity to a T cell receptor, or activate alymphocyte bearing a T cell receptor.

Antigen/HLA class I binding to a T cell, or activation of a T cell, canbe measured by any suitable method known in the art. Peptide-specificAPC-T cell binding and activation are measurable. For example, T cellengagement of antigen-presenting cells that express HLA-A2 reportedlycauses PIP2 to accumulate at the immunosynapse, whereas cross-linkingMHC class I molecules does not. See, Fooksman et al. (2009) CuttingEdge: Phosphatidylinositol 4,5-Bisphosphate Concentration at the APCSide of the Immunological Synapse Is Required for Effector T CellFunction, J. Immunol. 182:5179-5182.

Functional consequences of the interaction of a lymphocyte bearing aTCR, and a class I-expressing APC, are also measurable and include cellkilling by the lymphocyte. For example, contact points on the α2 subunitof HLA-A2 by CD8+ CTLs reportedly generate a signal for Fas-independentkilling. HLA-A2-expressing Jurkat cells apoptose when contacted (byantibodies) at epitopes on the HLA-A2 molecule known (fromcrystallographic studies) to contact CD8, without any apparent reliancean the cytoplasmic domain. See, Pettersen et al. (1998) The TCR-BindingRegion of the HLA Class I α2 Domain Signals Rapid Fas-Independent CellDeath: A Direct Pathway for T Cell-Mediated Killing of Target Cells? J.Immunol. 160:4343-4352. It has been postulated that the rapid killinginduced by HLA-A2 α2 contact with a CD8 of a CD8+ CTL may primarily bedue to this Fas-independent HLA-A2-mediated pathway (id.), asdistinguished from TCR-independent α3 domain-mediated killing—which byitself can induce apoptosis (see, Woodle et al. (1997) Anti-human classI MHC antibodies induce apoptosis by a pathway that is distinct from theFas antigen-mediated pathway, J. Immunol. 158:2156-2164).

The consequence of interaction between a T cell and an APC displaying apeptide in the context of MHC I can also be measured by a T cellproliferation assay. Alternatively, it can be determined by measuringcytokine release commonly associated with activation of immune response.In one embodiment, IFNγ ELISPOT can be used to monitor and quantify CD8+T cell activation.

As described herein, CD8+ T cell activation can be hampered in thegenetically modified non-human animals described herein due tospecies-specific binding of CD8 to MHC I. For embodiments where aspecies-specific CD8 interaction is desired, a cell of a geneticallymodified animal as described herein (e.g., a rodent, e.g., a mouse or arat) is exposed (e.g., in vitro) to a human cell, e.g., a humanCD8-bearing cell, e.g., a human T cell. In one embodiment, an MHC classI-expressing cell of a mouse as described herein is exposed in vitro toa T cell that comprises a human CD8 and a T cell receptor. In a specificembodiment, the T cell is a human T cell. In one embodiment, the MHCclass I-expressing cell of the mouse comprises a peptide bound to achimeric human/mouse MHC I or a humanized MHC I complex (which includeshuman β2 microglobulin), the T cell is a human T cell, and the abilityof the T cell to bind the peptide-displaying mouse cell is determined.In one embodiment, activation of the human T cell by thepeptide-displaying mouse cell is determined. In one embodiment, an invitro method for measuring activation of a human T cell by thepeptide-displaying cell is provided, comprising exposing a mouse or amouse cell as described herein to an antigen of interest, exposing acell from said mouse or said mouse cell (presumably bearing a peptidederived from the antigen in complex with human or humanized MHC I) to ahuman T cell, and measuring activation of the human T cell. In oneembodiment, the method is used to identify a T cell epitope of a humanpathogen or a human neoplasm. In one embodiment, the method is used toidentify an epitope for a vaccine.

In one embodiment, a method is provided for determining T cellactivation by a putative human therapeutic, comprising exposing agenetically modified animal as described herein to a putative humantherapeutic (or e.g., exposing a human or humanized MHC I-expressingcell of such an animal to a peptide sequence of the putativetherapeutic), exposing a cell of the genetically modified animal thatdisplays a human or humanized MHC I/peptide complex to a T cellcomprising a human T cell receptor and a CD8 capable of binding the cellof the genetically modified animal, and measuring activation of thehuman T cell that is induced by the peptide-displaying cell of thegenetically modified animal.

In various embodiments, a complex formed between a human or humanizedMHC class I-expressing cell from an animal as described herein is madewith a T cell that comprises a human CD8 sequence, e.g., a human T cell,or a T cell of a non-human animal that comprises a transgene thatencodes human CD8. Mice transgenic for human CD8 are known in the art.Tishon et al. (2000) Trangenic Mice Expressing Human HLA and CD8Molecules Generate HLA-Restricted Measles Virus Cytotoxic T Lymphocytesof the Same Specificity as Humans with Natural Measles Virus Infection,Virology 275(2):286-293; also, LaFace et al. (1995) Human CD8 TransgeneRegulation of HLA Recognition by Murine T Cells, J. Exp. Med.182:1315-1325.

In addition to the ability to identify antigens and antigen epitopesfrom human pathogens or neoplasms, the genetically modified animals ofthe invention can be used to identify autoantigens of relevance to humanautoimmune diseases, e.g., type I diabetes, multiple sclerosis, etc. Forexample, Takaki et al. ((2006) HLA-A*0201-Restricted T Cells fromHumanized NOD Mice Recognize Autoantigens of Potential ClinicalRelevance to Type 1 Diabetes, J. Immunol. 176:3257-65) describe theutility of NOD mice bearing HLA/β2 microglobulin monochain inidentifying type 1 diabetes autoantigens. Also, the genetically modifiedanimals of the invention can be used to study various aspects of humanautoimmune disease. As some polymorphic alleles of human MHC I are knownto be associated with development of certain diseases, e.g., autoimmunediseases (e.g., Graves' disease, myasthenia gravis, psoriasis, etc.; seeBakker et al. (2006) A high-resolution HLA and SNP haplotype map fordisease association studies in the extended human MHC, Nature Genetics38:1166-72 and Supplementary Information and International MHC andAutoimmunity Genetics Network (2009) Mapping of multiple susceptibilityvariants within the MHC region for 7 immune-mediated diseases, Proc.Natl. Acad. Sci. USA 106:18680-85, both incorporated herein byreference), a genetically modified animal of the invention comprising ahumanized MHC I locus including such an allele may be useful as anautoimmune disease model. In one embodiment, the disease allele isHLA-B27, and the disease is ankylosing spondylitis or reactivearthritis; thus, in one embodiment, the animal used for the study ofthese diseases comprises a human or humanized HLA-B27.

Other aspects of cellular immunity that involve MHC I complexes areknown in the art; therefore, genetically engineered non-human animalsdescribed herein can be used to study these aspects of immune biology.For instance, binding of TCR to MHC class I is modulated in vivo byadditional factors. Leukocyte immunoglobulin-like receptor subfamily Bmember (LILRB1. or LIR-1) is expressed on MHC Class 1-restricted CTLsand down-regulates T cell stimulation by binding a specific determinanton the α3 subunit of MHC class I molecules on APCs. Structural studiesshow that the binding site for LIR-1 and CD8 overlap, suggesting thatinhibitory LIR-1 competes with stimulatory CD8 for binding with MHCclass I molecules. Willcox et al. (2003) Crystal structure of HLA-A2bound to LIR-1, a host and viral major histocompatibility complexreceptor, Nature Immunology 4(9):913-919; also, Shirioshi et al. (2003)Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 competewith CD8 for MHC class I binding and bind preferentially to HLA-G, Proc.Natl. Acad. Sci. USA 100(15):8856-8861. LIR-1 transduces inhibitorysignals through its (intracellular) immunoreceptor tyrosine-basedinhibitory motif (ITIM). In NK cells, studies have shown that KIRs(inhibitory killer cell Ig-like receptors) lacking ITIMs (normallyincapable of inhibition) can inhibit in the presence of LIR-1(presumably through the LIR-1 ITIM) bound to the α3 domain of an MHCclass I molecule (see. Kirwin et al. (2005) Killer Cell Ig-LikeReceptor-Dependent Signaling by Ig-Like Transcript 2(ILT2/CD85j/LILRB1/LIR-1) J. Immunol. 175:5006-5015), suggestingcooperation between LIR-1 bound to MHC class I and KIRs and thus a rolefor HLA α3 domain binding in modulating NK cell inhibition.

As described above, MHC molecules interact with cells that do notexpress a TCR. Among these cells are NK cells. NK cells are cytotoxiclymphocytes (distinguished from CTLs, or cytotoxic T lymphocytes) thatplay a central role in the cellular immune response, and in particularinnate immunity. NK cells are the first line of defense against invadingmicroorganisms, viruses, and other non-self (e.g., tumor) entities. NKcells are activated or inhibited through surface receptors, and theyexpress CD8 but do not express TCRs. NK cells can interact with cellsthat express MHC class I, but interaction is through the CD8-binding α3domain rather than the TCR-binding, peptide-bearing α₁ and α₂ domains. Aprimary function of NK cells is to destroy cells that lack sufficientMHC class I surface protein.

Cross-linking MHC class I molecules on the surface of human naturalkiller (NK) cells results in intracellular tyrosine phosphorylation,migration of the MHC class I molecule from the immunosynapse, anddown-regulation of tumor cell killing. Rubio et al. (2004) Cross-linkingof MHC class I molecules on human NK cells inhibits NK cell function,segregates MHC I from the NK cell synapse, and induces intracellularphosphotyrosines, J. Leukocyte Biol. 76:116-124.

Another function of MHC class I in NK cells is apparently to preventself-killing. NK cells bear both activating receptor 2B4 and the 2B4ligand CD48; MHC class I appears to bind 2B4 and prevent its activationby CD48. Betser-Cohen (2010) The Association of MHC Class I Proteinswith the 2B4 Receptor Inhibits Self-Killing of Human NK Cells, J.Immunol, 184:2761-2768.

Thus, the genetically engineered non-human animals described herein canbe used to study these non-TCR or non-CTL mediated processes and todesign approaches for their modulation.

EXAMPLES

The invention will be further illustrated by the following nonlimitingexamples. These Examples are set forth to aid in the understanding ofthe invention but are not intended to, and should not be construed to,limit its scope in any way. The Examples do not include detaileddescriptions of conventional methods that would be well known to thoseof ordinary skill in the art (molecular cloning techniques, etc.).Unless indicated otherwise, parts are parts by weight, molecular weightis average molecular weight, temperature is indicated in Celsius, andpressure is at or near atmospheric.

Example 1 Construction and Characterization of Genetically ModifiedHLA-A2 Mice Example 1.1 Expression of HLA-A2/H-2K in MG87 Cells

A viral construct containing a chimeric HLA-A2/H-2K gene sequence (FIG.4A) was made using standard molecular cloning techniques known to askilled artisan in order to analyze chimeric human/mouse MHC Iexpression in transfected cells.

Briefly, a chimeric human HLA-A/mouse H-2K viral construct was madeusing the exon sequences encoding the α1, α2 and α3 domains of the achain and cloning them in frame with the mouse coding sequences for thetransmembrane and cytoplasmic domains from the H-2K gene (FIG. 4A,pMIG-HLA-A2/H2K). As illustrated in FIG. 4, the construct contained anIRES-GFP reporter sequence, which allowed for determining if theconstruct was able to express in cells upon transfection.

Viruses containing the chimeric construct described above were made andpropagated in human embryonic kidney 293 (293T) cells. 293T cells wereplated on 10 cm dishes and allowed to grow to 95% confluency. A DNAtransfection mixture was prepared with 25 μg of pMIG-HLA-A2/H2K,pMIG-human HLA-A2, or pMIG-humanized β2 microglobulin, and 5 μg of pMDG(envelope plasmid), 15 μg of pCL-Eco (packaging construct withoutpackaging signal Ψ). 1 mL of Opti-MEM (Invitrogen). Added to this 1 mLDNA mixture was 80 μL of Lipofectamine-2000 (Invitrogen) in 1 mL ofOpti-MEM, which was previously mixed together and allowed to incubate atroom temperature for 5 minutes. The Lipofectamine/DNA mixture wasallowed to incubate for an additional 20 minutes at room temperature,and then was added to 10 cm dishes, and the plates were incubated at 37°C. Media from the cells was collected after 24 hours and a fresh 10 mLof R10 (RPMI 1640+10% FBS) media was added to the cells. This mediaexchange was repeated twice. After a total of four days, the collectedmedia was pooled, centrifuged and passed through a sterile filter toremove cellular debris.

The propagated viruses made above were used to transduce MG87 (mousefibroblast) cells. MG87 cells from a single T-75 flask were washed oncewith PBS. 3 mL of 0.25% Trypsin EDTA was added to the cells and allowedto incubate at room temperature for three minutes. 7 mL of D10 (highglucose DMEM; 10% Fetal Bovine Serum) was added to the cells/trypsinmixture and transferred to a 15 mL tube to centrifuge at 1300 rpm forfive minutes. After centrifuging the cells, the media was aspirated andthe cells resuspended in 5 mL D10. Cells were counted and ˜3.0×10⁵ cellswere placed per well in a 6-well plate. pMIG-human HLA-A2 orpMIG-HLA-A2/H-2K either alone or with pMIG-humanized β2 microglobulinvirus were added to the wells, with non-transduced cells as a control.Cells were incubated at 37° C. with 5% CO₂ for 2 days. Cells wereprepared for FACS analysis (using anti-HLA-A2 antibody, clone BB7.2) forHLA-A2 expression with or without β2 microglobulin.

The graphs (FIG. 48), as well as the table summarizing the data obtainedfrom the graphs (FIG. 4C) demonstrate that co-transduction withhumanized β2 microglobulin increases the expression of human HLA-A2 orchimeric human/non-human HLA-A2/H-2K, as demonstrated by the shift ofcurves to the right.

Example 1.2 Engineering a Chimeric HLA-A2/H-2K Locus

The mouse H-2K gene was humanized in a single step by construction of aunique targeting vector from human and mouse bacterial artificialchromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Pat.No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineeringof the mouse genome coupled with high-resolution expression analysis.Nat. Biotech, 21(6): 652-659). DNA from mouse BAC clone RP23-173k21(Invitrogen) was modified by homologous recombination to replace thegenomic DNA encoding the α1, α2 and α3 domains of the mouse H-2K genewith human genomic DNA encoding the α1, α2 and α3 subunits of the humanHLA-A gene (FIG. 5).

Briefly, the genomic sequence encoding the mouse the α1, α2 and α3subunits of the H-2K gene is replaced with the human genomic DNAencoding the α1, α2 and α3 domains of the human HLA-A*0201 gene in asingle targeting event using a targeting vector comprising a hygromycincassette flanked by IoxP sites with a 5′ mouse homology arm containingsequence 5′ of the mouse H-2K locus including the 5′ untranslated region(UTR; 5′ homology arm is set forth in SEQ ID NO: 1) and a 3′ mousehomology arm containing genomic sequence 3′ of the mouse H-2K α3 codingsequence (3′ homology arm is set forth in SEQ ID NO: 2).

The final construct for targeting the endogenous H-2K gene locus from 5′to 3′ included (1) a 5′ homology arm containing ˜200 bp of mouse genomicsequence 5′ of the endogenous H-2K gene including the 5′UTR, (2) ˜1339bp of human genomic sequence including the HLA-A*0201 leader sequence,the HLA-A*0201 leader/α1 intron, the HLA-A*0201 α1 exon, the HLA-A*0201α1-α2 intron, the HLA-A*0201 α2 exon, ˜316 bp of the 5′ end of the α2-α3intron, (3) a 5′ IoxP site, (4) a hygromycin cassette, (5) a 3′ IoxPsite, (6) ˜580 bp of human genomic sequence including ˜304 bp of the 3′end of the α2-α3 intron, the HLA-A*0201 α3 exon, and (7) a 3′ homologyarm containing ˜200 bp of mouse genomic sequence including the intronbetween the mouse H-2K α3 and transmembrane coding sequences (see FIG. 5for schematic representation of the H-2K targeting vector). The sequenceof 149 nucleotides at the junction of the mouse/human sequences at the5′ of the targeting vector is set forth in SEQ ID NO: 3, and thesequence of 159 nucleotides at the junction of the human/mouse sequencesat the 3′ of the targeting vector is set forth in SEQ ID NO:4.Homologous recombination with this targeting vector created a modifiedmouse H-2K locus containing human genomic DNA encoding the α1, α2 and α3domains of the HLA-A*0201 gene operably linked to the endogenous mouseH-2K transmembrane and cytoplasmic domain coding sequences which, upontranslation, leads to the formation of a chimeric human/mouse MHC classI protein.

The targeted BAC DNA was used to electroporate mouse F1H4 ES cells tocreate modified ES cells for generating mice that express a chimeric MHCclass I protein on the surface of nucleated cells (e.g., T and Blymphocytes, macrophages, neutrophils). ES cells containing an insertionof human HLA sequences were identified by a quantitative TAQMAN™ assay.Specific primer sets and probes were designed for detecting insertion ofhuman HLA sequences and associated selection cassettes (gain of allele,GOA) and loss of endogenous mouse sequences (loss of allele, LOA). Table1 identifies the names and locations detected for each of the probesused in the quantitative PCR assays.

TABLE 1 Probes Used For Genotyping Region  SEQ Detected  ID Probe Assayby Probe Sequence NO HYG GOA Hygromycin  ACGAGCGGGT  5 cassetteTCGGCCCATT C 1665H1 GOA Human HLA-A2  AGTCCTTCAG  6 α2-α3 intronCCTCCACTCA GGTCAGG 1665H2 GOA Human HLA-A2  TACCACCAGT  7 α2 exonACGCCTACGA CGGCA 5112H2 GOA Human HLA-A2  ATCCTGTACC  8 α2-α3 intronAGAGAGTG

The selection cassette may be removed by methods known by the skilledartisan. For example, ES cells bearing the chimeric human/mouse MHCclass I locus may be transfected with a construct that expresses Cre inorder to remove the “foxed” hygromycin cassette introduced by theinsertion of the targeting construct containing human HLA-A*0201 genesequences (See FIG. 5). The hygromycin cassette may optionally beremoved by breeding to mice that express Cre recombinase. Optionally,the hygromycin cassette is retained in the mice.

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99). VELOCIMICE® (F0 mice fully derived from the donorES cell) independently bearing a chimeric MHC class I gene wereidentified by genotyping using a modification of allele assay(Valenzuela et al., supra) that detects the presence of the unique humanHLA-A*0201 gene sequences.

Example 1.3 In Vivo Expression of Chimeric HLA-A/H-2K in GeneticallyModified Mice

A heterozygous mouse carrying a genetically modified H-2K locus asdescribed in Example 1.2 was analyzed for expression of the chimericHLA-A/H-2K protein in the cells of the animal.

Blood was obtained separately from a wild-type and a HLA-A/H-2K chimericheterozygote (A2/H2K) mouse. Cells were stained for human HLA-A2 with aphycoerythrin-conjugated (PE) anti-HLA-A antibody, and exposed to anallophycocyanin-conjugated anti-H-2K^(b) antibody for one hour at 4° C.Cells were analyzed for expression by flow cytometry using antibodiesspecific for HLA-A and H-2K^(b). FIG. 6A shows the expression ofH-2K^(b) and HLA-A2 in the wild-type and chimeric heterozygote, withchimeric heterozygote expressing both proteins. FIG. 6B shows expressionof both the H-2K^(b) and the chimeric HLA-A2/H2K in the heterozygousmouse.

Example 2 Construction and Characterization of Genetically Modified β2Microglobulin Mice Example 2.1 Engineering a Humanized β2 MicroglobulinLocus

The mouse β2 microglobulin (β2m) gene was humanized in a single step byconstruction of a unique targeting vector from human and mouse bacterialartificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g.,U.S. Pat. No. 6,586,251 and Valenzuela et al., supra).

Briefly, a targeting vector was generated by bacterial homologousrecombination containing mouse β2m upstream and downstream homology armsfrom BAC clone 89C24 from the RPCI-23 library (Invitrogen). The mousehomology arms were engineered to flank a 2.8 kb human β2 m DNA fragmentextending from exon 2 to about 267 nucleotides downstream of non-codingexon 4 (FIG. 7). A drug selection cassette (neomycin) flanked byrecombinase recognition sites (e.g., IoxP sites) was engineered into thetargeting vector to allow for subsequent selection. The final targetingvector was linearized and electroporated into a F1H4 mouse ES cell line(Valenzuela et al., supra).

Targeted ES cell clones with drug cassette removed (by introduction ofCre recombinase) were introduced into an 8-cell stage mouse embryo bythe VELOCIMOUSE® method (see, e.g., U.S. Pat. No, 7.294,754 andPoueymirou et al., supra). VELOCIMICE® (F0 mice fully derived from thedonor ES cell) bearing the humanized μ2 m gene were identified byscreening for loss of mouse allele and gain of human allele using amodification of allele assay (Valenzuela et al., supra).

Example 2.2 Characterization of Humanized β2 Microglobulin Mice

Mice heterozygous for a humanized β2 microglobulin β2m) gene wereevaluated for expression using flow cytometry (FIGS. 8. and 9).

Briefly, blood was isolated from groups (n=4 per group) of wild type,humanized β2 m, humanized MHC (i.e., human HLA) class I, and doublehumanized β2m and MHC class I mice using techniques known in art. Theblood from each of the mice in each group was treated with ACK lysisbuffer (Lonza Walkersville) to eliminate red blood cells. Remainingcells were stained using fluorochrome conjugated anti-CD3 (17A2),anti-CD19 (1D3), anti-CD11b (M1170), anti-human HLA class I, andanti-human β2 microglobulin (2M2) antibodies. Flow cytometry wasperformed using BD-FACSCANTO™ (BD Biosciences).

Expression of human HLA class I was detected on cells from singlehumanized and double humanized animals, while expression of β2microglobulin was only detected on cells from double humanized mice(FIG. 8). Co-expression of human β2m and human HLA class I resulted inan increase of detectable amount of human HLA class I on the cellsurface compared to human HLA class I expression in the absence of humanβ2 m (FIG. 9; mean fluorescent intensity of 2370 versus 1387).

Example 3 Immune Response to Flu an Epstein-Barr Virus (EBV) PeptidesPresented by APCs from Genetically Modified Mice Expressing HLA-A2/H-2Kand Humanized β2 Microglobulin

PBMCs from several human donors were screened for both HLA-A2 expressionand their ability to mount a response to flu and EBV peptides. A singledonor was selected for subsequent experiments.

Human T cells are isolated from PBMCs of the selected donor usingnegative selection. Splenic non-T cells were isolated from a mouseheterozygous for a chimeric HLA-A2/H-2K and heterozygous for a humanizedβ2-microglobulin gene, and a wild-type mouse. About 50,000 splenic non-Tcells from the mice were added to an Elispot plate coated withanti-human IFNγ antibody. Flu peptide (10 micromolar) or a pool of EBVpeptides (5 micromolar each) was added. Poly IC was added at 25micrograms/well, and the wells were incubated for three hours at 37° C.at 5% CO₂. Human T cells (50,000) and anti-human CD28 were added to thesplenic non T cells and the peptides, and the wells were incubated for40 hours at 37° C. at 5% CO2, after which an IFNγ Elispot assay wasperformed.

As shown in FIG. 10, human T cells were able to mount a response to fluand EBV peptides when presented by mouse APCs that expressed thechimeric HLA-A2/H-2K and humanized β2 microglobulin on their surface.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Entire contents of all non-patent documents, patent applications andpatents cited throughout this application are incorporated by referenceherein in their entirety.

1.-48. (canceled)
 49. A non-human animal comprising in its genome: afirst nucleotide sequence encoding a chimeric human/non-human MHC Ipolypeptide, wherein a human portion of the chimeric polypeptidecomprises an extracellular domain of a human MHC I polypeptide; and asecond nucleotide sequence encoding a human or humanized P2microglobulin polypeptide, wherein the first nucleotide sequence islocated at an endogenous non-human MHC I locus, and the secondnucleotide sequence is located at an endogenous non-human β2microglobulin locus, and wherein the non-human animal expresses thechimeric human/non-human MHC I polypeptide and the human or humanized β2microglobulin polypeptide.
 50. The non-human animal of claim 49, whereinthe animal is a rodent, and wherein the rodent comprises in its genome:a first nucleotide sequence encoding a chimeric human/rodent MHC Ipolypeptide, wherein a human portion of the chimeric polypeptidecomprises an extracellular domain of a human MHC I polypeptide; and asecond nucleotide sequence encoding a human or humanized β2microglobulin polypeptide, wherein the first nucleotide sequence islocated at an endogenous rodent MHC I locus, and the second nucleotidesequence is located at an endogenous rodent β2 microglobulin locus, andwherein the rodent expresses the chimeric human/rodent MHC I polypeptideand the human or humanized β2 microglobulin polypeptide.
 51. The rodentof claim 50, wherein the rodent does not express an extracellular domainof an endogenous rodent MHC I polypeptide and a functional endogenousrodent β2 microglobulin polypeptide from their endogenous rodent loci.52. The rodent of claim 50, wherein the first nucleotide sequence isoperably linked to endogenous rodent MHC I regulatory elements, and thesecond nucleotide sequence is operably linked to endogenous rodent β2microglobulin regulatory elements.
 53. The rodent of claim 50, whereinthe rodent is a mouse.
 54. The mouse of claim 53, wherein the endogenousMHC I locus is a mouse H-2K locus.
 55. The rodent of claim 50, whereinthe human portion of the chimeric polypeptide comprises α1, α2, and α3domains of the human MHC I polypeptide.
 56. The rodent of claim 50,wherein a rodent portion of the chimeric human/rodent MHC I polypeptidecomprises cytoplasmic and transmembrane domains of a rodent MHC Ipolypeptide.
 57. The rodent of claim 50, wherein the human MHC Ipolypeptide is selected from HLA-A, HLA-B, and HLA-C.
 58. The rodent ofclaim 57, wherein the human MHC I polypeptide is a HLA-A polypeptide.59. The rodent of claim 50, wherein the second nucleotide sequencecomprises a nucleotide sequence set forth in exon 2 to exon 4 of a humanβ2 microglobulin gene.
 60. The rodent of claim 50, wherein the secondnucleotide sequence comprises nucleotide sequences set forth in exons 2,3, and 4 of a human β2 microglobulin gene. 61.-71. (canceled)